Essay: On the close relationship between speciation, inbreeding and recessive mutations. Etienne Joly, [email protected], Toulouse, September 2010 All the ideas developed in this essay are relatively simple, and most of them are related to many Foreword previously published works. So much work, however, This past year, 2009, was the Darwin year, celebrating has already been published on evolution and speciation the 200th anniversary of Charles Darwin's birth, and 150 that an autodidactic newcomer such as myself could years since the publication of his fabulous milestone not hope to read, let alone understand and remember all book, ‘The Origin of Species’ (to which I will the primary papers published previously on evolution subsequently refer to as ‘The Origin’). At the start of and speciation. If I have failed to acknowledge 2009, I was inhabited by a nagging ethical concern : how previous works developing ideas related to those put would humans deal with a situation where a group of forward here, the reader can be assured that this was individuals found themselves fertile among one another, not done maliciously but simply as a result of my but with limited fertility with the rest of the human race ? relative naivety on the subject. I do, however, hold the In other words, could speciation occur within the human firm conviction that, if some of the ideas developed in race ? This concern sprouted from the idea that this essay prove to be correct and relatively novel, it chromosomal rearrangements seemed to me like a very was only possible because of this naivety. probable initial step of a speciation process, since systematic survey of the human populations have actually Because, as a rule, I have adopted the principle of shown that such rearrangements are relatively frequent ( never citing a paper that I have not read, numerous frequency of the order of 1/1000, [1] ). Furthermore, times during the writing of this essay, I have found given the amazing success of the human race, having myself unable to decide what particular paper to cite as resulted in the huge numbers of human beings currently the appropriate original source of a particular concept living on our planet, and given the amazing propensity of or observation. Although I have tried to read as many nature to generate new species, I felt that the chances primary papers as I could rather than reviews, I found must be quite high that speciation could occur within the that I simply could not read everything. In addition, human population. most books, and also many papers were not available Most scientist concerned with evolution and to me in our institute's library or freely online (As speciation would probably not share those concerns another rule, I refuse to pay for online access, because I because the commonly held view is that speciation is firmly believe that all primary research papers should most often allopatric, i.e. it occurs when populations of be freely available to all). In such situations when I had individuals evolve separately from one another for a not managed to read the primary papers (for whatever sufficiently long time that they would no longer breed reason), I have very often chosen to cite the very efficiently with one another when they are reunited, comprehensive et quite recent reference book and the mobility of modern humans would be very "Speciation" by Coyne and Orr (2004), and to refer to effective at countering this type of phenomenon. it as ‘C&O’, with the indication of the appropriate Contrarily to these views, I believe that, if chapter or page number. reproductive barriers arise, they are basically Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 equivalent to other characters, and must therefore have The ideas presented here are relevant to many, if not been selected for. In other words, there must be most, subjects related to speciation and evolution. conditions where it becomes advantageous for groups Another difficulty I have encountered in the writing of of individuals to stop breeding with the ancestral this essay is that of keeping the discussion focalised on population. The main conclusion that I reach by the the main subject of this essay, i.e. that new species do reflections developed in this essay is that the process of not arise by splitting in separate branches, but by speciation is primarily driven by advantageous “budding” away from the ancestral population as a recessive mutations, and that those will, in turn result of inbreeding among a limited number of promote reproductive barriers, favouring inbreeding. individuals. As a solution, for discussing several issues This past year has seen the publication of a plethora that seemed either too technical or rather peripheral to of review articles on the subject of evolution and this main subject, I have chosen to treat those as speciation, which have allowed me to start catching up separate short pieces which are provided either as on these vast subjects, and to mature my reflections on footnotes, or as addenda at the end of this essay. the mechanisms involved in speciation. The reading of these reviews had also allowed me to confirm that More complex subjects, such as evolvability, the those ideas I have developed are in disagreement with origins of life and of sexual reproduction, group level the generally held views, i.e. that allopatric speciation selection and altruism would, however, each have is the most common and probable route for the warranted whole essays, and it was beyond my appearance of new species. capacities to cover all those issues simultaneously. I have thus had to put back dealing with those other issues to later dates.

28/09/10 1 Abstract: One of the important reasons for this failure was related to an issue to which he alluded to repeatedly in Whilst the principle of adaptive evolution is his book, which is that species are basically impossible unanimously recognised as being caused by the process to define. The main problem, which he acknowledged of natural selection favouring the survival and/or himself, and stays whole today, lies in the fuzzy limit reproduction of individuals having acquired new between species and varieties: “From these remarks it advantageous traits, a consensus has proven much will be seen that I look at the term species, as one harder to find regarding the actual origin of species. arbitrarily given for the sake of convenience to a set of Indeed, since speciation corresponds to the individuals closely resembling each other, and that it establishment of reproductive barriers, it is difficult to does not essentially differ from the term variety, which see how it could bring a selective advantage because it is given to less distinct and more fluctuating forms. The amounts to a restriction in the opportunities to breed term variety, again, in comparison with mere with as many and/or as diverse partners as possible. In individual differences, is also applied arbitrarily, and this regard, Darwin himself did not believe that for mere convenience sake.” (The Origin, p. 52 mid Ch reproductive barriers could be selected for, and today II). most evolutionary biologists still believe that One of the most important concepts that derives from speciation can only occur through a process of the work of Darwin is that the process of life is one of separation allowing two populations to diverge constant evolution, which explains why so few of the sufficiently to become infertile with one another. I do, life forms that occupied the earth 20 millions ago are however, take the view that, if so much speciation has still around today. The somewhat uncomfortable but occurred, and still occurs around us, it cannot be a inescapable conclusion from this is that the existence consequence of passive drift but must result from a of every single one of the millions of species that selection process, whereby it is advantageous for surround us, including ours, must also be transitory, groups of individuals to reproduce preferentially with and this probably contributes to the difficulty that one another and reduce their breeding with the rest of many humans have in accepting the theory of the population. evolution, in addition to the fact that it also brings In this essay, I propose a model whereby new species serious questions as to the existence of an almighty arise by “budding” from an ancestral stock, via a God. The processes of evolution and speciation are, process of inbreeding among small numbers of however, very slow ones, and the 5000 years of human individuals, driven by the occurrence of advantageous history1 do not amount to even a tick on the clock of recessive mutations. Since the phenotypes associated to evolutionary times, and to our human eyes, the stability such mutations can only be retained in the context of of the world thus appears as if it should stay the same inbreeding, it is the pressure of the ancestral stock for ever, and so with the species that occupy it. The which will promote additional reproductive barriers, fact that species are not stable entities, but in constant and ultimately result in complete separation of a new evolution is another factor that adds to the difficulty of species. I thus contend that the phenomenon of defining them. speciation would be driven by mutations resulting in Initially, species were recognised and defined by the advantageous loss of certain functions, whilst naturalists and palaeontologists mostly in relation to adaptive evolution would correspond to gains of their anatomical features, and it is on the basis of these

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 function that would, most of the time be dominant. features that Linnaeus opened the way to taxonomic A very important further advantage of inbreeding is classifications in the middle of the 18th century. that it reduces the accumulation of recessive mutations Regarding taxonomic definitions of species, dogs are a in genomes. A consequence of the model proposed is particularly telling example of the fact that, when that the existence of species would correspond to a considering species based on morphological traits, metastable equilibrium between inbreeding and certain organisms can differ greatly in their anatomy outbreeding, with excessive inbreeding promoting and still belong to the very same species. speciation, and excessive outbreeding resulting in It is some hundred years after Linnaeus, and well irreversible accumulation of recessive mutations that after Darwin and Wallace had laid down the principles could ultimately only lead to the species extinction. of natural selection, that the biological species concept emerged, which introduced the notion of the importance of fertility, and of the capacity to hybridize Introduction: in the definition of species. Today, the most popular definition of biological species is that proposed by Among the myriad of reviews and articles that have Ernst Mayr in 1942, as "groups of actually or been written about “The Origin of Species” by Charles potentially interbreeding natural populations, which Darwin, a very large proportion underlines the fact are reproductively isolated from other such groups". that, despite the title of his book, what Darwin established was the mechanism of adaptive evolution by the process natural selection, but that he failed to provide answers to the many questions that surround 1 i.e. since humans first started scribbling cuneiform the origin of species. signs or hieroglyphs

28/09/10 2 The first thing to underline in this definition is that traits, that lead to the definition of subtypes, species are not defined as standalone entities, but morphotypes, races, varieties, subspecies, species .... always in relation to other species (which provides One remarkable observation, however, is that inasmuch some rationale, albeit retroactive, to the fact that the as legions of well documented examples exist where singular of species is species and not specie, which those types of varieties have been generated and/or refers to coined money). The second important point documented, very few, if any, examples exist where about the definition of biological species is about the truly significant reproductive isolation has been difficulty of implementing it. Indeed, many closely witnessed under domestication, or even produced related species still show some degree of fertility with experimentally. We will therefore have to ask one another. For example, many species that do not ourselves what it is about natural conditions that differs detectably hybridize in the wild can produce perfectly so significantly from experimental conditions or fit and fertile offspring under experimental conditions. domestication. Furthermore, even if one was to set a threshold value for the degree of hybridisation between two separate The most prevalent view about speciation today is populations to consider them as separate species, the that geographical separation is the most likely degree of mixing of populations can vary greatly mechanism for the origin of species: independent depending on circumstances such as population adaptation to different environments will push the densities, or environmental fluctuations such as clarity evolution of the two populations sufficiently apart that of waters for certain fish that use visual clues to their offspring would be unfit because outbreeding recognise their own kin. between the two populations will result in the More recently, the amazingly fast progress in disruption of coadapted gene complexes. The term molecular biology has allowed geneticists to follow used to describe this type of speciation is allopatry, as and quantify the occurrence of gene flow between opposed to sympatry, where ancestral and descendant divergent populations, and this is often taken into species coexist in the same environment (or parapatry consideration when discussing whether two if they exist side by side, with a hybridisation zone in populations represent “good species” or not. On the between). If two populations having evolved separately subject of gene flow, one can, however, take the come back in contact later on, the intermediate slightly provocative stance that gene flow can never phenotype of their offspring could make them unfit for reach the absolute zero, which is related to the fact that either environment, and this would then provide the all organisms are based on the same genetic code. selective pressure for the selection of additional Indeed, there is more and more evidence accumulating reproductive barriers, in a process called reinforcement, about the prominence of horizontal gene transfer and often referred to as ‘the Wallace effect’. Indeed, between all sorts of organisms, mediated by varied the earliest promoter of the view that reinforcement mechanisms that can involve viruses, and particularly could occur under the pressure of natural selection was retroviruses, or possibly by incorporation of whole undoubtedly Alfred Wallace, who disagreed with organisms or just DNA. And transgenesis is another Darwin’s views that reproductive isolation could not recent progress of technology which reinforces the possibly result from natural selection: “The sterility of notion that "zero gene flow" is only a theoretical limit first crosses and of their hybrid progeny has not been towards which speciation can tend. acquired through natural selection” (The Origin,

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 Summary of Hybridism chapter). This point was a Considering the various difficulties one encounters in subject of written exchanges and arguments in private trying to define species, I will not engage in the correspondence between the two around 1858, 10 years somewhat sterile debate (excuse the bad pun ) of what after their joint communication to the Linnean Society constitutes ‘good species’, or rather of when two in July 1858, but Wallace formally published his views groups of animals can be considered as separate only in 1889, some twenty year later, in chapter VII of species. And even less in the consideration of whether his book called Darwinism. asexual organisms can be grouped into species. Rather, I will only engage in a reflection within the ‘biological On the subject of allopatry versus sympatry, I do take species concept’, as initially defined by Ernst Mayr. a very divergent view to that adopted by a majority of Furthermore, in considering only groups of organisms evolutionary biologists to this day. Rather, I choose to that reproduce sexually, I will focus on the follow Wallace’s path against Darwin’s in thinking that phenomenon of speciation. Indeed, although species natural selection plays a major role in the reproductive are well nigh impossible to define, one cannot dispute isolation that defines species, and I shall actually that speciation occurs, i.e. the fact that, starting from an venture some steps further than Wallace, and will ancestral population, some groups of animals will start advocate in the following pages that natural selection breeding more among one another than with the rest of can act on the very first stages of reproductive the population, and will progressively acquire a range isolation, and not just on reinforcement after of characters that sets them apart from the original divergence has taken place. Such views were also, but group. This, in fact, happens everywhere and all the temporarily, those of Theodozius Dobzhansky early in time around us, in wild and domestic species and is the his career [2], when he stated that " ...Occurence of reason for the appearance of particular characters, or hybridisation between races and species constitutes a

28/09/10 3 challenge to which they may respond by developing or offspring, which is directly related to the consideration strengthening isolating mechanisms that would make that the cost of sex is two-fold [3], as compared to hybridisation difficult or impossible". In other words, I asexual reproduction, where each offspring inherits all contend that, if there is so much speciation, i.e. genetic of the parent’s genome. But this factor of two is not mechanisms causing reproductive isolation evolving quite a completely accurate measurement. Indeed, if we everywhere, all the time, it must be because there can consider a hypothetical species with the most outbred be basic, fundamental selective advantages for population possible, each individual of that species will subgroups of individuals to breed preferentially among still be more genetically closely related to all the other one another, and reduce their capacity to hybridize with individuals of the same species than to any other the rest of the population. As will become clearer later individual of a closely related species. In other words, on, I adopt the point of view that, if species arise as a all individuals of a given species share more common result of selective pressures, then most events of ancestors than they do with those of a closely related speciation, even in their earliest steps, must take place species. Hence when they breed within their own as a result of the pressure of natural selection, and must species, individuals do share some significant level of therefore occur in settings of sympatry, or at least relatedness with their sexual partner compared with parapatry rather than allopatry since, under allopatric that of an individual of another species. So, even in a conditions, there can be no selective pressure to reduce completely outbred population, the cost of sex is not breeding with individuals one seldom encounters. quite as high as two. And the more closely related an But this is a very counter intuitive stance because individual is to it’s partner, the less that cost will be, saying that for a handful of individuals to breed for both of them. Consequently, any evolutionary step preferentially among one another rather than with the that will favour inbreeding rather than producing rest of the population is basically equivalent to offspring with more distantly related individuals, even advocating that inbreeding can bring on a selective of the same species, will thus reduce the cost of sex. advantage. And it is common knowledge to almost Although the notion that “selfing” is potentially everyone that inbreeding can be disastrously advantageous can be traced as far back as 1941 [4], and disadvantageous, and that hybrid vigour almost always despite the broad interest in the very insightful “selfish brings your direct descendants a selective advantage. gene” theory developed by Richard Dawkins [5], this intuitively obvious advantage of inbreeding actually I will, however, endeavour to demonstrate that seems to have received only very limited attention over inbreeding can have numerous advantages, particularly the years (outside of the notable concept of optimal in the long run, and that the selective advantages outbreeding developed by Bateson over 30 years ago brought about by inbreeding are the main driving force [6]. For one of the more recent papers on the subject, behind the phenomenon of speciation, whilst the short see Kokko and Ots [7] ). term advantages provided by outbreeding will result in an accumulation of recessive mutations that can 2) Inbreeding is necessary for the expression of represent a threat for the survival of species in the long advantageous recessive phenotypes. Evolutionary run. “progress” is often perceived as the acquisition of new functions, relying on mutations causing the appearance I ) Potential advantages of inbreeding of new genes that will, most of the time, be expressed

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 as dominant, or co-dominant traits. There are many We will hence start our reflection by asking ourselves cases, however, where mutations resulting in the loss what the advantages of inbreeding could be. If one of certain functions can be advantageous for carries out a simple literature search for the single individuals. For example, losing certain patterns of keyword “inbreeding” on a server such as Google colours can bring definite advantages, such as the scholar, one can rapidly identify tens of thousands of stripes of the African ancestor of zebras and horses. citations. Upon rapid examination, it is actually striking Those stripes were presumably very advantageous for to find that, in over 90% of those, the word inbreeding remaining inconspicuous to predators in the savannah, is systematically associated with either depression, cost but probably had the reverse effect for the early or avoidance, compared to only a handful of papers equidae that colonised more northern and greener where the potential benefits of inbreeding are actually latitudes and would later evolve into horses. As could being considered. There are, however, at least three already be suspected from the observations reported by ways that can indisputably bring an advantage to Darwin in the ‘Analogous Variations’ section of The inbreeding, and a fourth one which is more Origin, and later elegantly recounted by Stephen Jay contentious, but not necessarily less relevant to the Gould [8], crosses between various species of equidae, phenomenon of speciation, or at least to the welfare, and more specifically between zebras and horses, and hence survival, of species. reveal that the stripy phenotype is the dominant one. For the ancestors of horses to loose their stripes, 1) Reducing the cost of sex: The first and most significant inbreeding must therefore have occurred to undisputable advantage of inbreeding is that it reduces express that recessive stripe-less phenotype, and the cost of sex. Indeed, in sexual reproduction, each similar reasoning could be applied for the loss of any parent passes only half of its genome to each of its dominant character that may have been selected for in

28/09/10 4 ancestors, but was no longer beneficial, for whatever outbreak and spread of virulent pathogens (accidental reason (climate modification, colonisation, sexual changes in environmental factors such as global character that is no longer attractive …). modifications of climate conditions, meteorite impacts, or volcanic eruptions can also play important roles in Outside of the visible external phenotypes such as reducing population numbers). Under such conditions those considered in the previous paragraph, the of hardship, numbers will drop, and the surviving capacity to resist infections by pathogens is another population will be very likely to become fractionated type of recessive trait that I perceive as particularly into small isolated groups, resulting in relatively high likely to play a major role in the selection of relatively levels of inbreeding within each one of these groups. inbred sub-populations. Most pathogens, and in Under a scenario whereby an epidemic by a very particular viruses, do show high degrees of specificity virulent pathogen results in the decimation of a large for their hosts. This is due to the fact that pathogens population of mostly outbred individuals, the use particular receptors to penetrate the body and/or the subsequent generations will hence be much more cells of their hosts. Infections by harmful pathogens consanguineous, and this will provide the opportunity will therefore eliminate individuals expressing that for phenotypes due to recessive mutations causing receptor, and select for organisms able to resist resistance to that pathogen to surface, and those would invasion because they carry mutated receptors to which be endowed with a massive selective advantage. that pathogen can no longer bind. Such characters of Furthermore, inbreeding among resistant homozygous natural resistance are, however, usually recessive individuals will be even more favoured over the next because heterozygous individuals will still carry one generations because out-crossing with less related gene for a functional receptor, which will suffice to susceptible individuals will result in all the offspring render those individuals susceptible to invasion by that being susceptible to the decimating pathogen. pathogen. One particularly relevant example of this is From this type of reasoning, we can thus see how, in the case of humans carrying the CCR5-Δ32 mutation, addition to reducing the cost of sex, inbreeding can which, when homozygous, provides complete spontaneously promote the revelation of advantageous resistance to HIV infection, and an increased survival recessive characters. Increased inbreeding may not of a couple of years when heterozygous [9]. This very often arise spontaneously, but will result from delayed sickness would, incidentally, favour the conditions that promote a reduction of the effective spreading of HIV rather than be beneficial to the size of populations, such as colonisation or epidemics population, and thus bring a further advantage to the caused by very virulent pathogens, and the recessive homozygotes for the CCR5-Δ32 allele. The geographic nature of the characters that would be selected for distribution of the mutant CCR5-Δ32 allele does under those conditions would then provide the grounds suggest that this mutation arose several hundred years for reinforcing this inbreeding even further. Pushing ago in northern Europe, and it is hypothesized that it this concept even further, Chris Grobbelaar actually was probably selected for because it provided proposed, over twenty years ago, the interesting idea resistance to a pathogen different from HIV, because that a mechanism of crisis inbreeding would be the HIV epidemic only arose much later, in Africa [9]. advantageous, whereby situations of stress would result in a shift from sexual preferences towards inbreeding Although the pressure of a particular pathogen can [11].

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 provide a very definite advantage to those individuals that can resist infection by that pathogen, the fact that 3) Fighting Muller’s ratchet: A third advantage of this resistance will only be found in homozygotes inbreeding is that, for diploid organisms, it is the only would be a major hindrance for the spreading of that effective way to fight off the accumulation of recessive resistant allelic form to a whole population, but would deleterious mutations in their genomes. The notion that hugely favour particular subgroups where that allele mutations accumulate inexorably in genomes over the would be homozygous, which could only occur course of generations is commonly referred to as through inbreeding. The presence of individuals with Muller's ratchet [12]. Muller advocated that a major significant degrees of inbreeding within the population reason for the prominence of sexual reproduction does not, however, necessarily need to pre-exist the among all animal species was due to the need to infection by the pathogen, but could arise as a result of eliminate these mutations through genomic increased intensity of natural selection, such as an recombination. Muller, in his early work on epidemic, resulting in sparse populations that would Drosophila, had documented himself that most new then have no option but to breed with their close kin. mutations tended to have recessive phenotypes. When Of note, increased levels of inbreeding in the it came to persistence of those in the genome over population can also arise as a consequence of generations, however, he considered that all mutations colonisation [10]. Indeed, in times of affluence, sizes of were basically dominant, and that even the most populations may swell to large numbers, and recessive deleterious mutations must have some slight inbreeding coefficients will then be minimal. effect ( 2 to 5 % ) on reproductive fitness [13]. Those Subsequent times of hardship will, however, inevitably weakly deleterious mutations would therefore be follow, if only because high population densities are eliminated progressively over successive generations. known to promote both the rise of predators, and the Muller, however, carried out all of his work before the

28/09/10 5 discovery of the structure of DNA and of how genes consequence of inbreeding will be that the allelic worked. Although his arguments were clearly valid for frequency of recessive mutations will be lower in the weakly deleterious dominant mutations, we now know offspring than in their parents. For each mutation, the that new mutations will much more often result in the efficiency of the process is, however, remarkably low. disabling of a gene or of a protein’s particular function Indeed, in the case of a heterozygous breeding pair, the than in a change of function, and only very rarely in the allelic frequency for the mutated copy of the gene generation of a new function. But for most genes, a would only pass from 0.5 in the parents (each single valid copy is usually largely sufficient to fulfil heterozygote for the deleterious allele), to 0.33 in the their function, and most mut/WT heterozygotes show offspring (see box 1). But inbreeding is the only very little, if any, reduction in fitness compared to WT practical way for the members of a species with an homozygotes [14, 15]. Hence mutations will much obligatory diploid genome to cleanse their genomes off more frequently give rise to completely recessive the recessive mutations that will otherwise inexorably phenotypes, having absolutely no detectable effect on accumulate over successive generations. The reason the fitness of heterozygotes, and only rarely to why I have used the word “practical” in the previous dominant, or co-dominant traits. Inbreeding, by sentence is because of the bdelloid rotifers, the one promoting the conditions whereby recessive mutations undisputed example of asexual diploid organisms, that can find themselves in a homozygous state, will hence seem to have adopted an alternative strategy to sex to allow the expression of those deleterious effects cleanse their diploid genomes from recessive resulting from recessive mutations. As can be inferred mutations, but as discussed in addendum 1, it calls from work on laboratory strains of knock-out animals upon such extremes that it would be impractical for such as drosophila or transgenic mice, at least a third, most other organisms. Haploid organisms such as and maybe more than half of the mutations that result prokaryotes do not have this problem of keeping their in the invalidation of genes, such as those interrupting genome from accumulating deleterious mutations, an open reading frame, would actually be expected to because in haploids, all mutations are dominant, and be directly lethal, or to have such serious consequences deleterious ones will hence be eliminated very rapidly. that the homozygous bearer of such mutations would Multiple cases exist in nature of the use of a haploid probably not go on to breed under natural conditions of state by otherwise diploid eukaryotes, and in addendum selection. 2, I have developed three such examples that I find The adjective “inbred” has clear derogatory particularly eloquent i.e. the cases of organisms that go connotations when referring to human beings and the through haploid stages, of the sexual chromosomes and commonly held perception about inbreeding is that it of the endosymbiotic organelles. promotes degeneracy of the genome. Somewhat Diploid genomes must have contributed greatly to the ironically, inbreeding actually results in “improving” adaptive ‘explosion’ which took place among the genome, and the fact that inbreeding results in eukaryotes 1,5 billion years ago. The most important elimination of recessive deleterious mutations from the factor for this must have been the newfound tolerance population is actually well known, at least by animal or of organisms to new mutations that would have been plant breeders and scientists : the extent of inbreeding instantaneously deleterious in haploid organisms. depression decreases over successive generations of Conceivably, this may even have allowed the diploid inbreedin 2[16]. Via this type of phenomenon, the organisms to “lower their guard”, i.e. to reduce the

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 fidelity of the replication of their DNA, and favour mechanisms of recombination [17], thereby favouring 2 I owe the notion that inbreeding is bad for your the appearance of novel adaptive mutations, helping offspring, but good for their genomes, and hence for them in particular to combat pathogens more future generations, to a conversation I had several years efficiently, or to adapt to new environments. This view ago with my former colleague Geoff Butcher regarding is supported by the fact that the vast majority of the criticisable habit of certain scientists of using metazoans of today are obligatory diploids. The outbred rodents for their experiments on the grounds drawback of relying only on diploid genomes is that that those are usually healthier and fitter than inbred this also gives rise to the insidious type of Muller’s ones. This practice indeed introduces genetic ratchet I have just discussed, whereby recessive variability in the experimental samples, which can lead deleterious mutations can start accumulating silently in to results that are either too variable to be significant, the genome of outbred individuals. Without sex, the or even sometimes plain artefactual. On this subject, benefits of a diploid genome would, thus, be very short Geoff Butcher expressed the extremely wise point of lived, especially on the evolutionary time scale, and view that, if a scientist wants to work with very healthy genomes would ultimately reach a mutational rodents, he/she should be using F1 animals obtained meltdown [18]. But sex without inbreeding is fraught from crossing two separate inbred strains. Those types with even more insidious, and thus far greater dangers of animals all have strictly the same genetic that, as we will see, can ultimately lead to species background, and are indeed extremely healthy because extinction. they benefit from full swing hybrid vigour, and carry basically no recessive or partially recessive deleterious mutations.

28/09/10 6 Box 1: Comparing the effects of accumulation of recessive deleterious mutations in populations undergoing various degrees of inbreeding and with a theoretical completely outbred population.

Panel A : Mendelian laws predict that when a crossing occurs between two individuals heterozygous for a recessive deleterious mutation, allelic frequency for that mutation drops from 0.5 to 0.33.

Panel A : When breeding takes place between reduction of 0.25 in fertility. This value of 0.17 is two individuals each carrying one copy of a rather compatible with the various estimates of defective essential gene, one quarter of their the rate of spontaneous mutations, which are, for offspring will be either non viable, or very unfit humans, between 0.1 and 3 new deleterious because they will be homozygous for the mutation per genome per generation [19]. deleterious mutation. If the mutation is truly Although I realise that those figures are probably recessive, the other three quarters will be inaccurate for the additive effect of multiple perfectly viable, and two out of three among that genes, it was beyond my limited mathematical viable offspring will be heterozygous for the capacities to perform more precise calculations. I mutation. The allelic frequency of that deleterious am confident, however, that others will later find allele will hence pass from 0.5 in the parents to such calculations rather straightforward, and it 0.33 in the offspring, and the mutation load from will then be particularly interesting to evaluate 1 to 0.66 mutations per individual. what types of equilibriums are reached for As a rough estimate based on the simplistic case various mutations loads, various rates of of a single gene, one could therefore say that a mutations, and various effective sizes of rate of spontaneous mutation of 0.17 per population (i.e. various degrees of inbreeding). generation (0.5 – 0.33) will be compensated by a

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010

28/09/10 7 Panel B: Evaluation of the fertility as a function of mutation loads and inbreeding coefficients.

We have seen in the previous paragraph that, based on The theoretical fertility of breeding pairs in a population calculations for a single gene, a drop of 0.25 in fertility can be calculated as a function of M, the average number would keep up with the rate of 0.17 new deleterious of recessive mutations per individual ( i.e. the mutation recessive mutation per genome every six generations. The load) and of I, the overall average inbreeding coefficient figures would possibly be slightly different if one in that population ( i.e. the probability that a locus taken considered the additive effect of multiples recessive at random in the genome will be homozygous by descent, deleterious mutations affecting different genes, each with corresponding to half the average degree of consanguinity lower allelic frequencies, and clearly different with of parents). The average fertility will then be (1- I)M. The different mutation rates. The mutation rate of 0.17 per different coloured curves were calculated for the indicated generation cannot, however, be very far from reality since inbreeding coefficients, and we can see that fertilities only the most extreme estimates go from 0.1 to 3, and a start to be significantly affected for populations with decrease in fertility of 0.25 does not seem a completely inbreeding coefficients > 0.01, corresponding to parents unrealistic figure to keep up with new recessive mutations with degrees of consanguinity of 0.02, i.e. roughly that of occurring once every six generations. This does not, third or fourth cousins. however, mean that one in four newborns would come to In parallel, one can also evaluate the fertility of breeding the world with mental retardation or grievous physical pairs in an infinitely large population, where there is defects. Indeed, most recessive mutations that touch effectively no inbreeding (in real populations, the average essential genes would be expected to cause spontaneous fertility would actually be a factor of the two). For premature abortions at very early stages of pregnancy, mammals, if one estimates that roughly one third of genes and many even before they would be recognised as are essential, this would amount to a total of miscarriages. From this point of view, it is actually rather approximately 104 essential genes. If the mutation load in striking to note that, in modern humans, miscarriages the population is M, the probability of any locus being occur at a rate of somewhere between 10 and 40 %. 4 Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 mutated will be M/10 , and the probability of carrying Whilst the occurrence of these miscarriages is clearly also two mutated alleles of any given gene will be (M/104)2 = related to other factors such as the age and the health of M2.10-8 and hence the effect on fertility would be (1- the mother, these figures suggest that it is not M2.10-8 )10.000 overall since the threat applies for every unreasonable to envisage that the price to pay to fight single one of the 10.000 essential genes. This is Muller’s ratchet is that a fair proportion of the zygotes ( represented as the thick red curve on panel B, and we can say 20 to 30 %) will have to be lost to compensate for the see that, whilst the chance of carrying two inactivated occurrence of one new recessive mutation every six copies of the same gene remains extremely low for generations. And these figures also seem compatible with mutation loads below 20, it starts becoming quite what one sees in mice. Indeed, although mice can have as significant for mutation loads over 30, and fertility will many as 10 to 12 pups in a litter, inbred strains are much drop below 75% when genomes have accumulated, on less prolific, with litters often limited to 4 to 6 pups. average, over 50 recessive mutations. For populations When I have had to sacrifice pregnant female mice for harbouring levels of consanguinity superior to 0.02, the experiments on embryonic tissues, I have often been reduction in fertility is, as could be expected, much more struck by the proportion of aborted foetuses one can find sensitive to mutation load, and for a population with an in the uterus of a gestating female mouse, which is often inbreeding coefficient of 0.06, a drop of fertility to 75% near 50%. Thus, even in inbred mice in which the will occur with a mutation load between 5 and 6, but this inherited mutation load must be close to zero, the rate of figure climbs to nearly 30 mutations for an inbreeding abortions suggests that de novo recessive mutations occur coefficient of 0.01. at a rate that is probably superior to one in six zygotes, or one in six generations. ******** End of Box 1 ********

28/09/10 8 Indeed, DNA replication is far from being a perfectly mutations will keep accumulating, the mutation load will faithful process, and the rate of appearance of mutations inexorably increase. Even at the lowest rate of the range in the genomes of vertebrates is commonly recognised to envisaged above, i.e. one additional recessive mutation be of the order of 2.10-8 per nucleotide for every every ten generations, the mutation load will thus still generation, although the complete sequencing of the increase rather rapidly until, as proposed by Muller [13], whole genomes of a family of four suggests it may be it reaches an equilibrium where as many mutations are half as high [20]. For mammals, since their haploid eliminated at every generation than arise due to new genomes comprises roughly 3.109 base pairs, each spontaneous mutations. This process of elimination, diploid newborn will thus carry, on average, around 100 which correlates directly with infertility, will, obviously, nucleotides that will differ from those it should have be greatly dependant on the inbreeding coefficient, i.e. inherited from its parents if DNA replication were on the effective size of the population. In box 1, I have perfectly faithful, and DNA perfectly stable and tried to evaluate how the accumulation of recessive completely resistant to damages by radiation and mutations in a population can affect the fertility of chemicals. Among those mutations, the vast majority individuals as a function of the inbreeding coefficient in will be silent, but some will modify or inactivate gene that population. From rather simplistic calculations, I functions, and most of those will be deleterious, but conclude that, if the rate of accumulation of recessive recessive (somewhere between 3% and 1‰ of mutations, mutations is of the order of one every six generations, i.e. between 3 and 0.1 per individual per generation) this will be compensated by a drop in fertility of the [19]. Exceptionally, a mutation will lead to a different or order of 0.25. These figures, although rather speculative, new function, leading to a potentially adaptive character, seem to be compatible with the rates of spontaneous and most of those will be dominant or co-dominant abortions one sees in human and mice, of which a fair (somewhere between 10-4 and 10-5 for every new proportion (I would guess between one and two thirds) mutation, i.e. one in every one hundred to one thousand are probably due to genetic causes. As already individuals). underlined by Muller 60 years ago [13], the proportion of miscarriages due to genetic defects necessary to keep the In the long run, the phenomenon of evolution will be mutation load in a steady state will be principally based mostly on the acquisition of new characters, dependent on the rate with which new mutations appear corresponding to dominant mutations. But this can very in the genome at every generation. The process of easily be obscured by the much higher prevalence of outbreeding will indeed reduce the initial frequency at recessive mutations. This can be ascertained by the which recessive mutations are found on both copies of a repeated observations that the particular characters gene, but this advantage will only last for a while, until selected for in domestic species prove almost the mutation load has increased to levels where the systematically to be recessive against the phenotype of decrease in fertility due to mutations once again the wild stock.3 compensates for the rate at which they appear. The Even if DNA replication could be selected to become advantage of outbreeding is thus very short lived on the completely faithful, this would not be a solution, because evolutionary time scale. And, as mentioned earlier, I organisms have no choice but to evolve continuously in contend that it opens the door to a much greater threat. the face of natural selection, just like Lewis Carol’s Red Indeed, if a large population undergoes extensive Queen, who needs to keep running just to stay in the outbreeding for hundreds of generations, the equilibrium

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 same place, as famously underlined by Leigh van Valen will only be reached when each individual carries, on [21]. But because evolution is blind, and occurs only by average, several dozens of recessive mutations in its random mutations, in order to have a chance to see genome. If that population undergoes a sudden increase adaptive mutations arise, there will be no avoiding the in selective pressures, for example because of a novel hundred fold excess of deleterious mutations, which will pathogen, of competition with another species, of a need to be eliminated by natural selection. As alluded to recrudescence in predators or of changes in the natural earlier, most of those deleterious mutations will, environment, the effective size of that population will however, be perfectly recessive, i.e. they will have no shrink, and the inbreeding coefficient among the phenotype in heterozygotes. Hence, within a large out- survivors will consequently become very significant4. If breeding population, the chance that one individual will carry two copies of an inactivated gene will be very low. 4 But those will consequently be transmitted to half of the There is a rather counterintuitive possibility regarding a offspring, and over successive generations, since such potential advantage for the fact that, in times of harshness, increased inbreeding coefficient causes 3 individuals to become smaller. Indeed, smaller Since they did not know about Mendel’s laws, the individuals require less nutrients for their survival, and capacity of certain mutations in both pigeons and dogs to size is also well known to be inversely proportional to complement one another to restore a wild type phenotype population density. Hence, a rather intriguing possibility after many generations of ‘true’ breeding did contribute lies with the idea that, under conditions of increased greatly to confuse both Darwin and Wallace about the natural selection, small sizes caused by inbreeding durability of acquired recessive traits. depression may actually bring on a selective advantage in the struggle for survival.

28/09/10 9 we imagine that the mutation load in such a large makes it possible for the vast majority of cells to population had reached 40, and that the reduced numbers sacrifice themselves either directly by apoptosis, or by of individuals causes the inbreeding coefficient to raise differentiating into somatic cells that have absolutely no to 0.03 in the remaining population, this will result in hope of generating offspring, for the benefit of the very only 30 % of fit zygotes.. If we consider that this would few that will be destined to the germ line. Similarly, if a happen under conditions where natural selection would population is comprised of many small groups of be particularly harsh, the delayed cost of having avoided individuals that are more closely related to one another inbreeding for the short term benefits provided by than to the rest of the population, I firmly believe that it outbreeding may well, in the long run, play a major role then becomes possible for natural selection to favour the in the rapid extinction of that species, as well as reducing evolution of collaborative or altruistic behaviours, their capacity to colonise new environments (in the because, in the end, even if those behaviours do not section ‘convergence of character’ of The Origin, directly benefit the individuals that undertake those Darwin himself stated ‘When any species becomes very altruistic behaviours, the members of that group, and rare, close interbreeding will help to exterminate it’). In hence, on average, all the genes of the gene pool of that cases where there is a relatively sudden shift in the group, will fare better than those of the “group next pressures of natural selection, such as those caused by door” that may have stuck with strictly selfish natural catastrophes, or by a global change in the earth’s behaviours. On this subject, in 1871, Darwin himself temperature, the resulting shrinkage in effective made the following statement in his book “The Descent populations sizes would thus be expected to be less well of Man”: tolerated by the more prominent populations, i.e. It must not be forgotten that although a high standard probably those having taken full advantage of extensive of morality gives but a slight or no advantage to each outbreeding. Incidentally, such a mechanism would individual man and his children over the other men of provide an explanation for the phenomenon of the same tribe . . . an increase in the number of well- punctuated equilibrium proposed by Gould and Eldredge endowed men and an advancement in the standard of [22, 23]. In the face of Muller’s ratchet, as Muller morality will certainly give an immense advantage to one himself very rightly stated 60 years ago, “We cannot eat tribe over another. our cake today and have it tomorrow” [13]p150. Although, when one looks at natural populations, 4) Inbreeding promotes population fragmentation, scores of examples can be found in all the kingdoms of which can, in turn, promote collaborative or altruistic life where altruistic, or at least collaborative behaviours behaviour: have apparently been selected for, the questions linked to From the point of view of the ‘selfish gene’ hypothesis group level selection remain very contentious issues [5], individuals should always favour their own interests, today. I know of no better example of cooperative or at least those of closely related individuals [24, 25]. altruistic behaviour than that of the lowly slime mould, On the other hand, mathematical modelling has led Dictyostelium discoideum, and I contend that it is certain population biologists to conclude that group level promoted by the ability of single cells to colonise new selection cannot work, and that for any behavioural trait niches, resulting in fragmented populations. One of the to be selected, that trait must have a direct selective reasons for which I find the example of Dictyostelium advantage for the individual. This type of reasoning is, particularly telling is that it is not complicated by the

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 however, based on the assumption that populations intervention of sexual reproduction ( see addendum 3 for consist of large numbers of individuals breeding freely more details). with the rest of the population. But natural populations In some cases, speciation could conceptually are not like that. If we only look at the human correspond to the need for populations having developed population, although all individuals can theoretically cooperative/altruistic strategies to fend off more selfish breed with all those of the opposite sex with apparently invaders. The issue of altruism is, however, really a side equivalent efficiencies, we can see that the human race is issue to the main focus of this essay. All I wish to say structured in ethnies, races, types, families … and that here is that, for group level selection to evolve, it must be certain characters are more prominent in certain groups selected for, and this selection can only occur in of individuals than in the rest of the population. populations that are fragmented into small groups of In addition to the well recognised and very significant genetically inter-related individuals, or in other words, advantage of slowing down the spread of pathogens, by sub-groups undergoing more inbreeding than if the population fragmentation has the other, much less direct population was considered as a whole. The fact that and less obvious benefit of favouring the evolution of inbreeding can have the additional characteristic of altruistic behaviours, by making group-level selection providing a selective advantage at the levels of possible (see [26] for a recent review). On the subject of populations simply reinforces the view that inbreeding group selection, I chose to adopt the view that, in can and will occur and will not always be avoided. This fragmented populations, each group effectively becomes will result in structured populations, which will, in turn equivalent to a multi-cellular organism (see [27] for contribute to the phenomenon of speciation. recent views on organismality). In metazoans, the fact that all the cells share the very same genetic makeup

28/09/10 10 5) Disadvantages of inbreeding: For the sake of themselves rather than with the rest of the population, in fairness of argument, it seems necessary to others words with the ancestral stock? counterbalance our arguments here, and underline that inbreeding also has several very significant We have thus underlined how inbreeding can have disadvantages. Indeed, when starting from an outbred numerous advantages, and how systematic outbreeding is population, inbreeding depression will result in a high actually a strategy that is only advantageous in the short proportion of completely unfit offspring, and in most of run, but that can lead to great drawbacks in the long run. the offspring being less fit than those from outbred I now propose to follow the path laid out by Darwin in breeding pairs. Another consequence of excessive the title of his book, and to focus on the very origin of inbreeding is that, by reducing the gene pool available species, i.e. to try to imagine what initial genetic event for generating varied combinations of genotypes, it will could eventually lead to the separation of a subgroup of result in less diversity, and thus in a more limited individuals that breed preferentially among one another adaptability of the populations. Hence populations that rather than with the rest of the population. undergo excessive inbreeding will be less likely to I have chosen to use the word 'saeptation' to describe a develop new functions than large populations undergoing process based on a novel mutation that will promote the outbreeding, where new functions bringing selective interbreeding between individuals carrying that mutation advantages could rapidly spread to the whole population, rather than with the rest of the population. In other and could further combine with other advantageous words, saeptation will be the consequence of a mutation functions that will have arisen independently in other that will promote increased inbreeding within a group individuals. Inbreeding may thus result in a slower rate inheriting that mutation, and thus in a reduction of the of evolution. gene flow between this new group and its immediate This last argument does, however, need to be balanced ancestral stock. by two counter-arguments. First, as we have seen An alternative word would have been isolation, but I previously, advantageous traits are not necessarily find that this somehow implies complete separation and dominant, and those that are recessive can only come to is therefore too strong. Furthermore, isolation has been light under some level of inbreeding. Thus, although used very often in the past to refer to allopatric inbreeding will reduce the probability of dominant traits speciation after geographical isolation, and I am actually spreading to whole populations, it will increase the trying to demonstrate here that biological speciation frequency at which recessive traits appear, and since the probably occurs most often as a consequence of mutations causing such traits are much more frequent interactions between two groups of individuals, and not than those causing novel functions this may balance the via a stage of physical isolation. The word reinforcement effect of inbreeding on slowing evolution. Second, as has would have been another possible alternative, but it been recognised for a long time, the rate at which implies that some sort of divergence has already taken characters can become fixed in populations is inversely place, and I am actually trying to focus on the very first correlated to the size of those population [28]. By step, or steps, that will eventually lead to speciation, i.e. reducing the effective size of populations, the slower rate to the existence of inheritable reproductive barriers. In of evolution caused by inbreeding may thus also be this essay, I will therefore use the word saeptation to compensated. As we will see later on, I actually contend describe genetically based processes that induce that excessive inbreeding, leading to excessive separation of a group of individuals from their immediate

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 speciation, will consequently result in the shorter ancestors, and reinforcement to describe processes that lifespan of individual species. decrease the gene flow between groups that have already Another potential disadvantage of excessive inbreeding been more or less separated by another mechanism, such is that it could result is reductions in the levels of as saeptation. polymorphism in a population, by provoking what would effectively amount to repetitive bottlenecks. For jawed Lets us now envisage what type of mutation could vertebrates, which rely on polymorphism at the level of eventually lead to saeptation. This supposed mutation the MHC (Major Histocompatibility Complex) for will, one day, occur on one strand of DNA, and hence be fighting and eliminating infectious pathogens, this would present in one gamete, and go on to be present on one be expected to have particularly nefarious consequences. chromosome of all the cells of one individual. As we will see later, however, comparing MHC polymorphism between related species reveals that 1) One-step speciation: inbreeding, and speciation, can apparently take place In complete agreement with Darwin's views, in without losing healthy levels of polymorphism over the obligatory sexual species, it is very difficult to see how MHC region [29, 30], and presumably over most of the complete reproductive isolation could occur as a single genome. step because the individual that would carry such a mutation would find itself immediately incapable of breeding with it's own kin, and would thus not stand a II) Focusing our reflections on what the ORIGIN of very good chance of passing on its genes. Conceptually, species could be. Or how can it sometimes be beneficial a very extreme case may lie with the remote possibility for a few individuals to breed preferentially among of a mosaic individual harbouring a mutation having

28/09/10 11 occurred in the precursor of its germ line, in which case will greatly facilitate the fixation of an advantageous several individuals in it's offspring could carry the same recessive phenotype [28]. This isolation of a small dominant mutation resulting in complete infertility with relatively inbred group would hence result in reduction the members of the original population. The brothers and of the gene flow with the ancestral group because the sisters carrying that mutation would then find themselves adapted group would occupy a different territory. This with no other potential partners than their sibs, and would not, however, really represent a step of biological extreme inbreeding among those would potentially lead speciation, i.e. bona fide reproductive isolation, because to a new species. The only figure case where this would if one individual of that adapted group ended up among not result in an extreme bottleneck would be if the individuals of the ancestral stock, it would probably chimerical founding adult (probably a male) could mate breed with them very happily and efficiently, and the with multiple different partners (females), and thus defining stripe-less phenotype would be diluted and only generate a population of offspring with sufficient surface on very rare occasions5. But it would lay the diversity. The odds of this type of scenario do, however, grounds for the evolution of further isolating characters seem extremely low. because, in the context of their isolated group, it would An alternative scenario for one-step speciation is the be very disadvantageous for individuals to breed with case of chromosomal speciation, which are mostly found stripy partners from the ancestral pool since all off their in plants, where new species are formed either by offspring would then end up with the dreaded stripes on autopolyploidy (both parents from the same species), or their back, and thus be much more susceptible to allopolyploidy (parents from closely related, but separate becoming eliminated by predators. species), where non reduction during meiosis results in offspring that will be fit, and fertile with one another, but Consequently, if an additional mutation took place in a not with the parental species ( C&O, p321). Such events member of that adapted group that led to more effective of speciation, have, however, only been seen in the types reproduction with kin than with individuals not carrying of plants that regularly carry out either parthenogenesis, that second mutation, the inherent disadvantage of such a or self fertilisation, which amount to extreme inbreeding. mutation due to the reduction of fertility with the rest of The individuals of such species must therefore carry very the adapted group would be balanced by a very low mutation loads. In addition, as pointed out by Coyne significant advantage to its bearers because it would help & Orr ( p 344), this type of speciation would never be prevent that sub-group of individuals from being re- possible for dioecious organisms, i.e. those that have a invaded by the dominant but disadvantageous trait. This strict separation between males and females, usually preferential mating with kin would also amount to defined by sexual chromosomes. promoting further inbreeding. This may be further facilitated by the fact that, when populations have 2) Saeptation scenarios caused by a recessive mutation: previously gone through stages of significant inbreeding, For the vast majority of metazoans, successive steps of the cost of inbreeding depression is very much reduced progressive separation thus appear as more likely because most recessive deleterious mutations will have scenarios to reach speciation, and I will now explain why already been cleansed from the genome. On the other I think the most likely scenario involves a recessive hand, even in this context, inbreeding will retain its mutation as the very first step, i.e. the initial saeptation, advantageous side of reducing the two-fold cost of sex. which will end up promoting partial reproductive Hence, from the above reasoning, we see that, in the

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 isolation of its bearers. The first reason for this is that, as context of an outbred population, a mutation that simply already alluded to earlier, outside of silent mutations, results in promoting inbreeding will struggle to become new mutations will most frequently lead to loss of established because it would have many disadvantages to functions, and will usually be recessive, for the simple weigh against the advantage of reducing the cost of sex. reason that it is easier to undo something that works than But in the context of a group having undergone to create a new function from scratch. But, as we have significant inbreeding, the cost of further inbreeding seen in the previous section, a loss of function does not would be much lower, and under the selective pressure necessarily mean a selective disadvantage. of the persistent threat posed by invasion by the ancestral Let us go back to the example of the horse precursors, stock, the probability of additional steps of saeptation and how they could have lost the stripes carried by their within that group would thus be much higher. zebra-like ancestors. In the first place, to reveal the non- striped recessive phenotype, some significant inbreeding 3) Scenarios involving two mutations (Dobzhansky- must have taken place. That inbreeding could actually Muller model ): To explain how mutations promoting have been promoted by the very fact that the group for reproductive isolation could ever appear in natural which the stripe-less phenotype was advantageous was in populations, Bateson (1909), Dobzhansky (1936) and the process of colonising more northern latitudes. Muller (1942) all came up with a similar hypothetical Colonising populations, having smaller effective sizes, have consequently higher inbreeding coefficients [10], 5 and we will see later that this is particularly relevant for As we will see later on, this type of phenomenon the situations of island colonisation. Another actually happens in sticklebacks, which gain a selective consequence of the small size of such a group is that it advantage by losing their armour plates when they colonise freshwater environments.

28/09/10 12 model, which is nowadays unjustly referred to as the that selective pressures would be driving the isolation, Dobzhansky-Muller model (see C&O, p 269). This rather than rely on chance for the separate evolution of model calls upon the existence of two completely two traits that will, at a later stage, turn out to be separated groups (allopatry), where two separate incompatible. One of the predictions inferred from the mutations take place that would each have no effect on Dobzhansky-Muller model is that the rate of the reproductive fitness in the group in which they arise, accumulation of reproductive barriers should increase but that would result in incompatibility between the with time, the so called “snowball effect” [31-33]. But groups if and when those two groups are brought back in this prediction does not actually allow to discriminate contact with one another. Such models are, however, not with the sympatric scenario described above. Indeed, if in line with Darwin’s views that each step along the very the threat of hybridisation is maintained throughout the long path of an evolutionary process must carry its own speciation process, one would expect a similar snowball selective advantage. In the context of a group carrying a effect: once some degree of reproductive isolation has recessive advantageous mutation, however, we can see started accumulating between the two populations, how the pressure of the outside populations, carrying resulting in reduced inclusive fitness of the hybrids dominant but disadvantageous mutations, could promote further than the simple initial loss of the recessive the selection of a mutation favouring reproductive advantageous phenotype (for various reasons including isolation from the ancestral stock. At the end of the reduced fertility, poor health or even lethality), the cost previous paragraph, I have argued that this selective of mating and/or breeding with the ancestral stock will pressure may be sufficient to promote further steps of be further increased. Consequently, the pressure for saeptation, i.e. isolation from the other members within selecting further mechanisms of reproductive isolation the adapted group, because the disadvantages of this will also be increased, and one would thus expect the rate mutation promoting inbreeding would be overcome by at which such traits are selected to go up, until such the advantage of resisting invasion by the dominant times when the two populations are sufficiently isolated disadvantageous phenotype. And this modified tilt of the that neither represents a significant threat for the other balance would be further favoured by the reduced one. inbreeding depression resulting from the relatively high level of inbreeding already present within that group. 4) Scenarios involving a dominant mutation : Lets us Another scenario is, however, possible, which is to a now consider whether a scenario can be envisaged certain degree related to the Dobzhansky-Muller model whereby a dominant mutation would promote saeptation. in that it would involve multiple steps, but those would The most obvious type of such a mutation would seem to occur in sequence, and not independently: the secondary be one that modifies the actual niche of the population, a steps of isolation would target traits specific to the phenomenon often referred to as ecological speciation. saeptated population which could quite possibly be the Indeed, if individuals carrying a novel mutation can start one having driven the saeptation, but not necessarily. occupying new territories (geographical, seasonal, Indeed, during the initial phases of saeptation, inbreeding nutritional ...) they will, in this new territory, naturally among a limited number of individuals would result in a find themselves in the presence of those other individuals high proportion of other genes becoming homozygous, carrying the same mutation, which will, by definition, be and could thus reveal additional recessive phenotypes descended from the same ancestor, and will therefore be only rarely encountered in the ancestral population. In their close relatives (sibs or cousins). Since we are now

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 addition, in other genes than the one having driven the talking about a dominant mutation, to allow the first saeptation, certain alleles would have become much individuals with the new mutation to find mates to more frequent, either because they were genetically reproduce, the initial separation between the adapted linked to the advantageous mutation, or simply because subgroup and the ancestral stock can, however, only be the smaller size of the population had favoured their drift partial, and the possibility of hybridisation between the towards fixation. For these three types of genes two groups must therefore be preserved. Although (additional recessive phenotype, genetically linked to the inbreeding among colonisers may carry an initial cost advantageous recessive mutation, gene having reached because of inbreeding depression, this could easily be fixation by chance), the allelic frequencies would offset by the advantage of the lack of competition in the therefore be very different in the saeptated inbred new territory, and the inbreeding depression would only population than it the ancestral one. And those would be transient, and recede after a few generations. then represent as many potential targets for the selection Although, as we will see later, dominant mutations could of isolating mechanisms that would prevent the play important roles in further steps of the speciation individuals of the group from mating back with the process, i.e. in reinforcement, it is thus hard to envisage ancestral group. Technically speaking, this would, how they could, on their own, promote the selection of however, not represent saeptation, but reinforcement, reproductive barriers with the ancestral stock. In the case because the mechanism of isolation would specifically of a dominant mutation leading to the colonisation of a target the outsiders, and not the direct ancestral stock, i.e. new niche, however, the increased inbreeding among the the isolated group. This type of scenario would thus individuals carrying the mutation would, however, involve two or more steps like the Dobzhansky-Muller greatly increase the probability of revealing some model, but the fundamental difference with that model is additional recessive characters, of which some may turn

28/09/10 13 out to be adaptive to the newly colonised environment. Importantly, whether the mutation driving the And those recessive mutations could, in turn, provide the saeptation is completely recessive or co-recessive could grounds for a selective advantage to stop breeding with have significant consequences on the size of founder the ancestral stock. populations. Indeed, in the case of completely recessive mutations, those could stay completely silent for long 5) The special cases of periods of time within a population, and hence surface when crossings occur between individuals that are not 5a) Co-dominant and co-recessive characters. necessarily very closely related to one another. In the When crossings occur between parents each carrying case of a co-dominant mutation, however, the new homozygous but different alleles of a gene, this will intermediate character will be expressed in half the often result in the offspring harbouring intermediate offspring of the founding individual, and the founding phenotypes. Co-dominant characters will usually be due population will thus necessarily be comprised mostly by to a mutation that changes the function of a gene, and the brother-sister matings, or close cousins at best. We will heterozygous offspring will hence express both come back later to considerations regarding the size of functions. Co-recessive mutations are due to mutations founder populations and preservation of heterogeneity in that inactivate a gene, but because of gene dosage the population. effects, the offspring expresses less protein than the parent with the functional gene (this is, for example, 5b) Chromosomal translocations. often seen for pigments). If a mutation that changes the Chromosomes can be either circular, as in most function of a gene that is also subject to gene dosage, the bacteria and in endosymbiont organelles, or linear, as in resulting phenotypes will be at the same time co- all eukaryotes and a few bacteria. As far as I know, there dominant and co-recessive. Within the frame of the are no known organisms with circular chromosomes that analyses carried out in the previous paragraphs, co- can carry out meiotic sexual reproduction, and all recessive mutations would seem particularly prone to eukaryotes also have multiple chromosomes. Multiple promoting speciation. Indeed, if such a mutation brings linear chromosomes thus appear as a prerequisite to about an adaptive phenotype, such that the partial gain or meiosis, with three chromosomes being the smallest the partial loss of a function makes it possible to colonise number documented, in the fission yeast a new niche (warmer or colder climates, higher altitude, Schizosaccharomyces pombe (most species have several different food, different breeding time…), the dozens, and up to several hundreds, or even over one heterozygotes of the first few generations would be thousand in certain ferns). One of the main reasons closely related to one another, but would be expressing having driven the arrangement of the genetic information intermediate phenotypes that would not separate them on such multiple and linear structures is almost certainly too much from the ancestral stock, and hence allow for to promote one of the main purposes of sex, i.e. to the generation of multiple individuals. Crossing of those achieve an efficient shuffling of the genes between semi-adapted individuals with one another would be individuals having evolved in parallel, via both inter- and favoured by the fact that they would occupy that new intra-chromosomal recombination. Another commonly niche. This would result in a quarter of their offspring recognised advantage of this arrangement in metazoans becoming homozygous for the adaptive trait, which they (multi-cellular organisms) is that the maintenance of would hence express more strongly, and would possibly telomeres provides a certain level of safeguard against

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 be restricted to occupying only the newly colonised the rogue selfish multiplication of cells that will lead to niche, with little or no possibility of contact with the cancer. Outside of these two obvious advantages, I ancestral one. The intermediate phenotype of the perceive that the arrangement of genomes on multiple heterozygotes could thus be likened to some sort of linear chromosomes is also likely to play a central role in stepping stone for the assembly of an isolated, the phenomenon of speciation. Indeed, in line with the necessarily more inbred group of individuals observation that even closely related species almost homozygous for the adaptive trait. Once that group has always differ in their chromosomal architecture, the role been constituted, in addition to the fact that the cost of of chromosomal rearrangements in speciation has long sex would be higher with the outside group than within been hypothesized (see C&O p 256-267, citing White the group, a further advantage would be that additional 1978). One hurdle to this hypothesis, however, is that a adaptations to the new niche would probably be selected chromosomal rearrangement such as the textbook for quite rapidly, and the phenotype of the offspring that example of a whole arm reciprocal translocation pictured would result from encounters with the ancestral stock in fig. 1, will result in a significant decrease of the would very possibly make them unfit for either fertility of the individuals in which this translocation environment. This would thus provide the grounds for occurs in the first place, with half of the zygotes the Wallace effect, i.e. for the selection of further predicted to be non viable when mating occurs with mutations reinforcing the reproductive isolation between individuals of the rest of the population, which would not the two populations. We can thus see how co-recessive carry this translocation. Once the translocation has traits could conceptually promote reproductive isolation become fixed within a group, complete fertility will be even more rapidly than completely recessive ones. restored to all individuals of that group. But for this to happen, heterozygous individuals carrying the same

28/09/10 14 mutation will first have to mate with one another, and Although other types of chromosomal remodelling, such under such circumstances, the proportion of viable as inversions or centromeric fusions, may not affect the offspring is predicted to drop even a little bit more, from proportion of viable offspring to the same extent as 1/2 to 3/8 ( Fig. 1), and this is without accounting for the reciprocal translocations, some effect on the proportion inbreeding depression that would necessarily occur since of viable gametes would still be expected since such those individuals would, logically, have to be closely modifications are known to disturb the phenomenon of related to one another. Furthermore, the translocation chromosomal pairing that takes place during meiosis would then become homozygous in only 1/6 of their [34]. viable offspring ( corresponding to 1/16 of the zygotes).

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 Given the above considerations, it is difficult to see as very likely candidates for the promotion of such how chromosomal translocations could ever take hold in events of speciation associated to chromosomal any population and reach fixation unless they were rearrangements. Indeed, their expression is known to be directly associated with a phenotype endowed with a strongly influenced by their genomic environment, and very significant selective advantage. This is, however, since they are involved in regulating many embryonic not so difficult to envisage. The most direct way would and ontogenic processes, minor changes in their be if one of the breakpoints involved in the remodelling expression patterns could often have very significant resulted in the disruption of a gene, leading to a effects on the shape and aspect of organisms. In the beneficial recessive mutation of the type discussed in the process of speciation, events of chromosomal previous section. Alternatively, the effect could be less remodelling could thus conceptually fulfil two roles direct: it is now well known that, for many genes, the simultaneously, i.e. a phenotypic alteration leading to genomic context plays an important role in their some selective or adaptive advantage, coupled to a regulation [35], and it thus does not seem unlikely that reduction of the fertility with the ancestral stock. certain chromosomal alterations could have a direct In addition to the argument that even very closely dominant effect on one or more of the genes surrounding related species usually do show significant differences in the breakpoints where the chromosomal alteration took their chromosomal architecture, the view that place. This would be expected to potentially provoke chromosomal remodelling plays a significant role in significant phenotypic alterations, and those modified speciation is also supported by the relatively high phenotypes may, sometimes, be advantageous, or frequency at which chromosomal rearrangements do adaptive to novel environments. Homeotic genes appear occur, and could thus conceivably be sufficiently

28/09/10 15 frequent to occur even in small isolated groups flow between the two groups, but would actually further undergoing saeptation. Indeed, systematic studies of increase the threat because the hybrid offspring would be human karyotypes have revealed that detectable neo- viable, but less fertile. rearrangements occur at a frequency of approximately After an initial step of saeptation, further steps of one in a thousand [36]. Whilst many of such reproductive isolation from the ancestral stock would rearrangements may result in spontaneous abortions (as therefore be clearly advantageous for that new, but much many as 50 % of human reproductive failures could be smaller group. Within the saeptated group, any further due to chromosomal abnormalities), many others will be mutation that would increase reproductive isolation from viable, as testified by the fact that as many as one in 625 the ancestral stock would therefore be expected to carry a phenotypically healthy human beings carries a reciprocal very significant advantage, and could thus rapidly spread chromosomal translocation [37]. Because those to the whole group, which the small size of the saeptated translocations do provoke significantly reduced fertility, group would further favour. unless they are linked to an advantageous phenotype, We can now ask ourselves what sort of mutations they are expected to get progressively eliminated from and/or traits could intervene in the progressive large outbreeding populations over successive establishment of completely separated populations, i.e. generations. But finding them at such a sizeable undetectable gene flow, such as what one witnesses frequency vouches for the fact that individuals carrying between closely related groups recognised as separate chromosomal rearrangements will occur quite often in species, although living side by side in natural humans, and hence probably in all species. environments. Once a saeptated group has been In addendum 4, I discuss how chromosomal constituted, in which individuals are all more closely rearrangements could promote saeptation, leading to related to one another than to the rest of the ancestral speciation, depending on whether they would be group, further steps of reproductive isolation will not associated to dominant, recessive, or co-dominant/co- necessarily have to rely on recessive mutations. In the recessive advantageous traits, and I reach the conclusion previous paragraphs, I have argued that, in some that the most likely scenario would seem to be those circumstances, the selective pressure from the ancestral involving co-dominant phenotypes. Although stock may be sufficient to promote further steps of chromosomal rearrangements associated to strictly saeptation within the isolated group, based on additional recessive advantageous phenotypes are also conceivable, recessive mutations, which would be favoured by the and would lead to very rapid fixation of the increased inbreeding coefficient, and consequent low rearrangements, this would seem an unlikely occurrence, mutation load within that saeptated group. On the other and would provoke very tight bottlenecks for the founder hand, dominant traits would presumably spread to the populations. group very rapidly, and would have the added advantage Another possibility to consider is that chromosomal that it would not require the elimination of the rest of the rearrangements could be selected for as secondary group. In the long run, as long as hybridisation with the saeptation steps, i.e. simply because they would reduce ancestral stock remains a threat, any additional trait that fertility of a saeptated group when they breed with the significantly reduces the chance of producing offspring ancestral group, even if it would initially also involve with members of that ancestral population could bring on some reduced fertility with the rest of that founder group. a sufficient advantage to be selected for. As such, Conceptually, this decrease in fertility may sometimes mechanisms that prevent either mating or the formation

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 represent a sufficient advantage to be selected for its own of zygotes (and hence called prezygotic isolation) such sake, as suggested by the observation that chromosomal as sexual preference, occupation of niches more remote rearrangements are more frequent between sympatric from the ancestor, gamete incompatibility or even than between allopatric species of drosophila [38]. The culturally acquired traits could all contribute to recessive beneficial advantage would then be one of protecting the newly formed group from the threat of maintaining optimised fertility, but the process would breeding with the ancestral population. This type of certainly be much more direct, and thus favoured if the reasoning, which assumes an asymmetric relationship chromosomal translocation was directly associated to a between a newly formed group and a more numerous mutated gene leading to an advantageous phenotype. ancestral stock, provides an explanation for the observation first underlined by Muller in 1942 that 5c) The Wallace effect: Secondary steps towards incompatibilities between closely related species are very speciation, i.e. reinforcement . often asymmetric (C&O, p274). Once a small group of individuals has ‘sprouted’ from the ancestral stock, if they have to keep expressing the When prezygotic isolation is not complete, and closely recessive advantageous traits that drove the constitution related species can still mate and produce zygotes, those of that group, breeding with the ancestral stock will hybrids are often found to be either non-viable, or fit, but represent a permanent threat for the welfare of their sterile. Scenarios for the development of this type of offspring, and the different sizes of the two groups will barrier between species, which is called postzygotic be a factor that greatly increases the weight of this threat. isolation, are slightly more difficult to envisage because If the initial mutation was directly linked to a one needs to explain how it can be more advantageous chromosomal rearrangement, this would limit the gene not to produce offspring at all than to produce hybrids.

28/09/10 16 For explaining this, however, I find one observation Similarly to what was discussed above, those genes, particularly useful: whilst problems of viability usually whether carried by autosomes or sexual affect offspring of both sexes, problems of sterility chromosomes, would thus represent potential targets usually follow Haldane’s rule, and almost always affect for the selection of new mutations carried by the only the heterogametic sex ( C&O, p311-312). We can sexual chromosomes: newly mutated genes would thus consider the problems of explaining hybrid lethality still function well with the genotypes frequently and hybrid sterility as completely separate. present in the isolated group, but would no longer Regarding hybrid lethality, I can see two obvious work in combination with the genotypes prominent reasons whereby it would be better not to produce in the ancestral stock. This would be particularly offspring at all than to produce hybrids. First, if there is a likely for the heterogametic sex because any significant cost to one or both parents for the rearing of mutation carried by one or the other of the sex offspring that will ultimately be unfit, it will be chromosomes, even those corresponding to a loss of advantageous to save those resources for the subsequent function, would be immediately dominant, as rearing of “purebred” offspring. And second, if the already underlined by Muller in 1940, and hybrid offspring occupies a niche that overlaps with that formalised as the dominance theory put forward by of the purebred offspring, those two types of offspring Turelli and Orr [39]. Since sexual chromosomes are, would then be competing with one another. Sometimes, a necessarily, endowed with many genes related to further threat for the more inbred offspring could lie with sexual reproduction, a likely phenotype resulting the fact that the hybrids would be particularly fierce from such a selective process would be one affecting competitors because they would benefit from hybrid the sexual capacities, and hence result in the sterility vigour, and it would thus be best not to produce that of the heterogametic sex. Alternatively, the genes hybrid offspring at all. involved in the reproductive isolation may be part of the large number of genes carried by the Regarding the phenomenon of hybrid sterility, I can see chromosomes which are diploid in half the three ways whereby it can be promoted, which are not individuals (X in mammals and flies or Z in certain mutually exclusive. insects, fish, reptiles and birds. For the sake of 1) The first one lies with chromosomal clarity and simplicity, I will use X as an example for rearrangements. As already mentioned in the previous the rest of this paragraph, but I could just as well pages, chromosomal rearrangements are very often have used Z). Lets us now envisage that a mutation associated to phenomena of speciation, and even takes place on a gene carried by the X chromosome, closely related species are often found to diverge by such that the gene product will still function well several chromosomal structural differences. Although with the allelic form of some other gene found at hybrids carrying a single chromosomal translocation high frequency in the saeptated group threatened by such as the one depicted on figure 1 will only see their hybridisation, but will no longer function with the fertility drop by 50 % when they mate with allelic form(s) found in the ancestral group. As long homozygous individuals of either type, this proportion as the individuals of the group breed among one will drop further for every additional chromosomal another, that mutation would have no detectable rearrangement and soon reach figures approaching effect, and would thus not really have any reason to zero. A factor further contributing to sterility is the spread to the whole group. But if hybridisation with

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 observation that chromosome pairing has been found to the ancestral stock took place, because this mutation be a necessary step for the proper completion of corresponds to a loss of function, it will most of the meiosis, at least in eutherian mammals ( C&O p 262- time result in a recessive phenotype, and it would 264, citing Searle1993 ). As we have seen in the thus have the typical characteristics of X-linked previous pages, the fixation of such rearrangements deficiencies, i.e. be silent in diploid female would be most likely to occur when they are directly offspring, and expressed in the hemizygous males. linked to an advantageous phenotype. The observation The X chromosome carries many genes involved in that there are more differences in chromosomal vital functions, and disabling of those would architecture between drosophila species living in presumably result in lethal phenotypes. Under the sympatry that in allopatry [38] does, however, suggest threat of generating hybrid offspring with an outside that the reduced fertility provided by such group, the individuals carrying such mutations rearrangements may sometimes represent a sufficient would then be endowed with a definite advantage advantage per se. that would explain how, although neutral within the saeptated population, such mutations could be 2) The second reason lies with the haploid nature driven to fixation in the group undergoing of the sex chromosomes in the heterogametic sex speciation. The above scenarios would thus explain (see addendum 2). As already discussed earlier why phenotypes of reproductive isolation are often (section II-3 ), following a process of saeptation, the asymmetric, why they are often stronger in allelic frequencies of many genes in the newly situations of sympatry, and provide potential formed group would be expected to be significantly explanations for Haldane’s rule, i.e. why, when different from that in the ancestral population. inter-species crosses take place, if only one sex is

28/09/10 17 affected, it is usually the heterogametic one that is populations that have evolved side by side to adapt to either non-viable [40], which I contend could often coastal or mountainous conditions than between those occur by recessive mutations of vital genes on the X that have evolved on separate islands [41]. chromosome, or sterile, by mutations of genes This is also exactly what happens with domesticated involved in sexual reproduction carried either by the species. Under conditions of domestication, species can Y or the X chromosome. diverge to become very noticeably different, and reproduce for scores of generations under very divergent 3) The third reason for which hybrid sterility may conditions of selection, yet they do not become infertile be selected for lies with the fact that sexual with one another. In this regard, I find the example of reproduction is usually much more costly for dog breeds particularly telling. Upon comparing the females than for males, with the latter having the remains of a great Dane and of a Chihuahua, or of a capacity to produce virtually unlimited numbers of Dachshund and a Saint-Bernard, no taxonomist in their offspring. In the case where a population undergoing right mind would ever place them as belonging to the speciation competes with the ancestral stock for the same species. Yet, when my steps take me to public occupation of a niche, I contend that the generation parks or other places where people go to let their four of hybrids where females are fit and fertile, but legged friends relieve their natural needs, I am often males are unfit can represent an extremely struck (and amused) to see how dogs of very different advantageous strategy. These aspects will be sizes and appearances can still recognise one another as developed further in section IV. potential sexual partners. And we do know that they do indeed belong to the same species. They all share exactly III) There is probably seldom such a thing as truly the same chromosomal architecture as wild wolves. In allopatric speciation : fact, if all these dogs of different sizes were placed in a In the previous section, we have seen how giant enclosure and fed regularly, some sexual advantageous recessive traits could promote the preferences between certain types may surface (see long formation of small saeptated groups within large citation of Wallace’s book in section V), pregnancies populations, and how the need to keep expressing those between small females and large males may turn out to recessive phenotypes could subsequently drive be fatal for the mothers, and the smaller males would reinforcement, i.e. further steps of reproductive isolation, probably not fare too well in fights with larger ones, but based on a whole array of different mechanisms. The in the end, all those dogs would produce extremely fit recurring theme of the reasoning developed in the offspring that would certainly be much more previous pages is that reproductive isolation would not homogenous than the starting population, and would arise passively, but as a result of selection under the almost certainly contain genes inherited both from the pressure of an outside group, most frequently the Chihuahuas and the great Danes. I contend that, if ancestral population. Even if today, the majority of domesticated species do not undergo speciation, it is evolutionary scientists believe that most events of because the process of selection is carried out by the speciation must have occurred in allopatry, I do actually breeders, and not by natural selection, where individuals, believe that if truly allopatric speciation ever happens, and groups of individuals, directly compete with one i.e. for whole populations to drift apart sufficiently to another for the production of offspring and the become infertile with one another, it must be an occupation of a niche, and loosing this competition

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 extremely slow process, and consequently a very rare means dying with no offspring. occurrence. Indeed, if populations of individuals are In settings of domestication, even if most characters completely separated, there will be no selective pressure that are selected by the breeders are recessive, and could for evolving features that will further reduce gene flow even be sometimes be associated to chromosomal between the two groups, because the gene flow will rearrangements, there is never any direct pressure for already be non extant. If the geographical barrier is later individuals to stop breeding with the ancestral stock, and lifted, the features of the individuals in each group will there can thus be no selection for either saeptation, or almost certainly be quite different because they will have reinforcement. The fact that different domestic breeds, adapted to their respective environment. Some including dogs and pigeons, have now been maintained mechanisms of preference between similar phenotypes in complete separation from one another for hundreds of may favour reproduction among the individuals having generations without any discernible sign of speciation co-evolved, but since there will have been no selective ever being witnessed is, in my eyes, one of the stronger pressure, I contend that there would be no reason why arguments against the possibility that passive speciation, the individuals from either group should have become resulting from drift, could play a significant role in the infertile with those of the other group. This is in fact in phenomena of speciation that are clearly taking place complete agreement with what has been very recently continuously in the natural world. described for Caribbean Anoles lizards. Those have evolved independently for millions of years on separate Another argument against the role of intrinsic genetic islands that only joined relatively recently to form the incompatibility resulting from a random process in the large island of Martinique, and more reproductive evolution of reproductive isolation can be found in barriers appear to have been selected for between comparing the estimations of lifetime of species, and of

28/09/10 18 the time it takes for such incompatibilities to develop. resulting reduced fitness of the individuals may, Indeed, for both mammals and birds, the fossil record however, be well tolerated because, in the newly tells us that the average time of existence of a species is colonised territories, those few individuals will have no around one million years [21], whereas the time it takes competition from kin, and presumably very few for the genomes of mammals to diverge sufficiently to predators and pathogens adapted to them. Because of this become genetically incompatible is estimated to be initial episode of inbreeding, however, the cost of around 2-4 million years [42], and well over 10 million subsequent inbreeding will be expected to become much years for birds [43]. Given those numbers, one would reduced after just a few generations, and this population thus have to envisage that, if genome divergence by drift of colonisers would then presumably multiply quite was the main factor responsible for speciation, most rapidly to occupy its newfound niche. But the characters species would become extinct before they would have a of the ancestral stock would probably not be best adapted chance of evolving into another species. to their new environment, and conditions would thus seem very favourable for the selection of new characters Detractors of the views expressed in this essay would allowing them to adapt. As we have seen before, not fail to point out that there are many documented mutations leading to recessive characters are much more examples of allopatric speciation, i.e. where groups of frequent than dominant ones. And these would be even individuals that were geographically separated have more likely to come to light in the envisaged conditions, become “good species”, i.e. completely infertile with one where inbreeding would be favoured both by the small another. But to counter this argument, we only need to size of the population, and by the fact that inbreeding think back to the ancestral species, the one which is depression would be minimal. Hence, if a recessive presumed to have occupied the ancestral territory, and mutation occurred that brings on an adaptive advantage colonised the new one (or, as proposed by Darwin, to the new environment of the colonised island, there become split in two by a rising mountain range). If the would be a very significant advantage for the individuals two modern species cannot breed with one another, then carrying the adapted, recessive, phenotype, to reduce we can safely assume that at least one of the two would their breeding with the rest of the colonising group. Any also have been infertile with the ancestral species. But, mutation coming to reinforce that saeptation would thus by definition, individuals of that ancestral species were be advantageous, and would not necessarily have to be initially present on the two territories, and that species recessive itself. Hence, mechanisms reinforcing the cannot have disappeared before the appearance of a isolation of the adapted group from the rest of the subgroup of individuals that were less fertile with the population, such as traits of genetic or post-natally ancestral individuals, and would eventually lead to the inherited sexual preference, gametic incompatibility, modern species. The logical consequence of this point of genomic incompatibility or chromosomal rearrangements view is that, when allopatric speciation appears to have could evolve within that group, whereas the initial occurred, it actually probably corresponds to several selection of such traits is normally not favoured in larger, successive steps of 'sympatric' saeptation, with the new, more outbred populations, where inbreeding depression better adapted group replacing the ancestral intermediate. is high. The most striking examples of speciation often occur The picture we get from the above scenario is one on islands, and when Charles Darwin visited the where, when a secluded niche, such as an island, is Galapagos in the course of his voyage on the Beagle, the initially invaded by very few individuals, successive

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 observation of all the very unusual specimen found on steps of saeptation and/or reinforcement among a few those remote islands would later on help him greatly to adapted individuals will be greatly favoured by the initial formulate his theory of evolution, as well as to consider inbreeding episode. And at every step, the better-adapted the idea that geographic isolation could contribute to descendants of that group would most probably wipe out speciation because of the independent evolution of the less-well adapted stock of their immediate ancestors. populations that would progressively become infertile For every one of these steps, the reduction of gene flow with one another. with the immediate ancestors would not necessarily be Let us now consider the phenomenon of island very high but, although that ancestral stock would have speciation from the point of view developed in the long been eliminated from the island, each one of those previous paragraphs, i.e. that speciation occurs mostly as steps reducing the fertility between the population of a consequence of natural selection, in other words in a adapted individuals and their immediate ancestors, and context where it is advantageous for subgroups of consequently would be expected to have a cumulative individuals to stop breeding with the ancestral stock. effect on the fertility between the adapted population and Colonisation of islands are, inherently, very rare events, the ancestral stock. Hence, if the population of and even more so for an obligatory sexual species individuals that have adapted to the island through because this implies that at least two individuals from successive steps of saeptation and/or reinforcement was opposite sexes find themselves on the same island at the ever brought back in contact with the more numerous, same time, which could, quite often, be brothers and outbreeding population which stayed on the continent, sisters descended from a single pregnant female. The individuals from those two groups would very probably initial population will, consequently, go through a very be completely infertile with one another, even if the tight bottleneck, with extreme degrees of inbreeding. The latter one had not evolved away much from the ancestral

28/09/10 19 stock. The speciation process so witnessed would, however, not really have occurred in allopatry, but as a To conclude this section, I would say that, for most succession of sympatric steps which can only occur cases considered as undisputable examples of allopatric under the selective pressure of the immediate ancestral speciation, the times of separation are often much longer stock. An argument that supports the validity of this type than the expected lifetime of the species considered. of reasoning is the recurrent observation that events of Also, since in most cases ancestor and speciating groups speciation seem especially prone to occur in the context probably co-exist for much less time than the lifetime of of small populations, such as those promoted by small species, it is not surprising that so few cases of speciation islands. The size of the niche itself (for example a small appear sympatric. But it is not because we do not see it island, or a small lake) could indeed be the main factor happen that sympatric speciation does not happen. Thus, contributing to the maintenance of a relatively high contrarily to the stance proposed by Coyne and Orr, I degree of inbreeding, and hence to the reduced level of contend that allopatric speciation should not be inbreeding depression that can promote speciation. Thus, considered as the default mode (C&O, p84). Rather, to even in the context of islands that are not completely prove that truly allopatric speciation has ever taken isolated from the regular invasion by individuals from place, I advocate that one would have to demonstrate that the mainland (such as the Baleares, the Caribbean or the no step of saeptation has taken place during the Canaries), or from other nearby islands (such as the evolutionary process, whereby one sub-population would Galapagos), small islands have been found to be have become reproductively isolated from its immediate particularly propitious to speciation in all sorts of genera sympatric ancestor, and subsequently eliminated it. (birds, lizards, mammals, insects…).

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010

28/09/10 20 IV) What relationship can be expected between the ancestral stock. Under such conditions, one would thus different modes of speciation, the mechanisms of expect reinforcement, or further saeptation, to be selected reproductive isolation that are being selected for, and for essentially in the younger group. the diversity of the newly separated population?6 Particularly interesting examples of parapatric Despite the arguments presented in the previous speciation are those provided by ring species, whereby section, there is no denying that the conditions under new species arise in successive steps around a which speciation occurs (sympatry, parapatry, allopatry) circumventable geographic barrier such as a mountain would be likely to play important roles on both what (Greenish Warbler around the Himalaya), an ocean types of reproductive isolation mechanisms are being (Herring Gulls around the Atlantic Ocean) or a valley selected for, and on the size and diversity of the founding (Ensatina Salamanders around the central valley in population that will ultimately result from the speciation California) [45]. In the end, although some gene flow process. In figure 2, I have drawn simplistic sketches that persists between direct neighbours, i.e. between ancestral would correspond to scenarios of speciation occurring in stock and new populations having colonised a new those three conditions. In this drawing, the shapes parapatric niche, the species that end up meeting at the represent the niche occupied by a population. I feel that opposite end of the ring are completely infertile with one an important point to underline regarding the nature of another. The simplest explanation for this type of niches is that they are not solely linked to geographical phenomenon seems to be that the additive effect of constraints, but to many other factors such as the nature incomplete reproductive barriers will finally result in of the nutrients, the timing of the life cycle, the identity truly isolated species. With regard to the ideas proposed of other partner species such as pollinators for plants, or here, it will be particularly interesting to see if characters hosts for parasites, etc… All in all, I perceive that the can be identified that have contributed to the progressive defining point between parapatry and sympatry is adaptation of the species along the barriers, and when whether the niches of two populations undergoing those are due to recessive characters, whether this is speciation are sufficiently non overlapping that neither accompanied by more significant reproductive isolation could ever wipe out the other one. On the other hand, from the ancestral stock. even if two groups have such different life styles or life cycles that they seldom breed with one another, but still In a context of sympatric speciation, the younger group compete for the very same food, or for the same territory, having undergone saeptation will have to compete one could fully expect that one of the two protagonists directly with the individuals of the ancestral stock for the will, sooner or later, inherit a new character allowing it occupation of the niche. Whilst the speciating group to eliminate the other one completely. In short, when would have the advantage of the newly acquired, but occupation of the niche equates to competition for recessive, advantageous trait such as the resistance to a survival, I will call this sympatry; if the two populations pathogen, the ancestral group would have the important can exist side by side without one ever being wiped out advantage of a much more numerous starting population, by the other one, I will call this parapatry; and when the presumably harbouring more diversity. The two populations have so few interactions that neither is a counterbalance of this would be, however, that this larger threat for the other one, I will call this allopatry. and older group would probably also carry a heavier In figure 2, within the niches, I have not represented mutation load than the speciating group. In the context of populations as uniform entities, but as fragmented in a competitive struggle between the two groups,

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 subpopulations, where the less intense areas correspond population densities would presumably thin out for both to reduced densities of population, and hence higher groups, leading to increased inbreeding. Whilst this degrees of inbreeding. Under conditions of parapatric would not be a problem for the younger group, it would speciation, the group undergoing speciation will colonise most probably result in a very significant drop in fertility a different, adjacent niche (new territory, different for the older and more numerous ancestral stock because nutrients, different breeding period …). For the reasons it would carry a heavier mutation load. In such exposed in section II, speciation will be initiated if the circumstances, because of both the newly acquired character that allows this colonisation is recessive, and selective advantage having driven the saeptation, and its hybridisation between the two groups would thus lighter mutation load, the odds would thus seem very represent a much bigger threat for the members of the likely to tilt towards the younger population most of the newly formed and less numerous group than for the times. At later stages, with the increased scale of its progressive elimination, the intensity of the threat would, 6 however, bear more heavily on the ancestral stock, and it The views developed in this section are somewhat would thus be expected that mechanisms of isolation related to the considerations on founder effects could be selected for in groups of animals derived from developed as models of ‘genetic revolution’ by Mayr the ancestral stock. If those corresponded to recessive (1954) (see C&O p 387-393), ‘founder-flush theory’ by phenotypes, leading to saeptation, this may actually Carson (1975) and ‘genetic transilience’ by Templeton result in the selection of a separate isolated group [44] , but contrarily to those, I do not believe that drift (figured by a pink spot), which would then have to under conditions of true allopatry would suffice to compete both with the first speciating group and with the promote the fixation of characters of reproductive ancestral stock. In the end, this could result in the isolation other than on extremely rare occasions.

28/09/10 21 ancestral group having been replaced by two separate behaviours, because a sterile hybrid female would daughter species. effectively neuter the sexual activity of her fertile male The lower part of figure 2 sketches the scenario of partner. In this regard, it is quite remarkable to note that, island colonisation developed in the previous section, whilst 90% of bird species are monogamous, only an whereby a handful of founding individuals give rise to a estimated 3% of mammals are7. iii) An important completely isolated population, and the high inbreeding consequence of the process of ‘sleeping with the enemy’ conditions, resulting in low mutation load, favour will be that, among the offspring resulting from crosses successive steps of sympatric saeptation that will between the purebred stock and the hybrid homogametic ultimately result in complete infertility between the offspring, 50 % will become homozygous for the population occupying the island and the ancestral stock. advantageous recessive trait, and could thus formally The common point between the last two scenarios of join the saeptated group. Through this type of process, speciation is that the newly formed groups have to the saeptated group, which may initially have been compete with their direct ancestors for the occupation of endowed with rather limited genetic diversity, may thus the niche. In line with Darwin’s views, the stakes in this progressively incorporate a significant portion of the struggle are ‘the survival of the fittest’, which implies the diversity present in the ancestral stock. ultimate elimination of the other kind. Hence, for a population undergoing sympatric speciation, to This last point brings us to consider the question of the paraphrase General Philip Sheridan, “the only good evolution of genetic diversity through the process of ancestor is a dead ancestor”. For achieving this, I speciation. In this regard, great insights can be gathered perceive that post zygotic mechanisms, which will often from comparing the diversity of the major affect only the heterogametic sex, are particularly histocompatibilty complex (MHC) between closely effective strategies which can have, as we will see, related species. The MHC, which is found in all jawed multiple types of advantages. Indeed, in the context of a vertebrates, is the most polymorphic region of their newly formed group, even if the members of the group genomes. The reason for this is that it is involved in are somewhat fitter because of the advantageous many aspects of immunity, and thus under very strong recessive character they express, they may also be selection, with the diversity of MHC molecules being affected by more inbreeding depression, and the much used to fight off the amazing capacity of pathogens to smaller effectives of the newly founded population could adapt to their host. Comparisons of allelic diversity easily be overwhelmed by the sheer number of the between closely related species such as human and competitors. Lets us now envisage the consequences of chimpanzee [29], or mouse and rat [30], have revealed generating hybrid offspring where one sex is fertile and that certain polymorphisms of MHC molecules have the other one is either sterile or dead. For the next survived all the successive steps of speciation that have generation, this will result in a deficit in potential separated each species from their common ancestor. partners of the heterogametic sex, and that situation will Such observations thus strongly suggest that speciation, have several consequences: i) it will free up some space even if it involves inbreeding, does not necessarily have in the niche that the purebred members of the saeptating to occur via very tight bottlenecks, and thus tend to group can then move into without competition. In a support the validity of the types of scenarios proposed at further elaboration, one could even envisage that there the end of the last paragraph. could be an advantage to the sterile hybrids being very fit

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 because of hybrid vigour. They would thus occupy a In the case of human and chimps, the presumed last large portion of the niche, but would eventually die with common ancestor is called Nakalipithecus, who lived no offspring, and leave all that space vacant for the some 10 million years ago. Since then, although the offspring of their fertile neighbours. ii) In mammals and precise details of our ancestry are stilled hotly debated, it flies, where the males are heterogametic, a further is clear that our family tree must have counted at least advantage would be conferred by the fact that the males half a dozen successive species, first belonging to the can produce offspring with numerous partners at very gender Australopithecus ( anamensis, afarensis, africanus little cost. In conditions where hybrid females remain …), and then to the gender Homo ( habilis, erectus …). fertile and hybrid males are sterile, the males from the Over that time, 30 million sequence differences have saeptated group would thus find themselves with more accumulated between the human and chimp genomes, potential partners. Subsequently, the offspring resulting corresponding to 1% divergence, as well a 10 from mating with those hybrid females would generate more fertile females, and, if the sterility was due to only 7 one locus, presumably only 50 % of fertile males. On the subject of bird monogamy, in The Origin, Although this type of reasoning could also apply to Darwin himself underlines several times the fact that it species where the females are heterogametic (certain has been possible to derive and keep so many different insects, fish, reptiles and birds), this effect of the process breeds of pigeons because those can be paired for life, would be somehow restricted by the fact that females and then kept in the same aviary. His report of the are, by nature, restricted in the number of eggs, and common observation of sudden reversion of certain hence offspring that they can generate. This could phenotypes towards wild type phenotypes does, however, be compensated for by monogamous however, vouch for the fact that even among birds, some adultery still occurs regularly.

28/09/10 22 chromosomal modifications (9 inversions and 1 size of the region of reduced diversity should therefore centromeric fusion ), of which one can reasonably expect be much more limited than in the case of the selection for that about half must have taken place in the branch a recessive trait. leading to humans. Incidentally, it is interesting to note The prediction that follows this reasoning is that this that the number of chromosomal rearrangements is may actually provide the means to identify the regions roughly of the same order as the number of speciation carrying the genes involved in events of speciation, and steps on the presumed path between Nakalipithecus and conceivably even the very genes having driven the the two modern species. This does not, however, prove speciation9, as well as a reasonably accurate estimation in any way that each of those rearrangements was of the dates at which it happened. Indeed, if one necessarily correlated to a phenomenon of speciation. documented the levels of diversity of silent intergenic Indeed, some of those chromosomal rearrangements DNA over the whole genome for a good number of could have become fixed in the population because they unrelated individuals belonging to the same species, this were directly linked to dominant beneficial characters. would not only provide the means to really evaluate the When two separate human genomic sequences are degree of inbreeding within a population, as well as the compared, one allegedly finds around 0.2% divergence, inbreeding coefficient for each individual, but one would which would amount to 6 million mutations per haploid also expect to be able to rapidly identify regions of genome. Our species has only been around for 250.000 limited diversity. Although the occurrence of years, and thus approximately 10.000 generations. As we chromosomal rearrangements may confuse the have seen previously, new mutations accumulate at the interpretation [34, 46], the gene responsible for driving rate of approximately 60 per haploid genome per the fixation would be expected to be at the centre of such generation. One would thus expect only 600.000 new regions, and the level of divergence of intergenic mutations to have accumulated in each genome since the sequences within those regions would provide a appearance of Homo Sapiens. The level of divergence relatively precise estimate of the time of fixation. seen between human genomic sequences thus provides additional support for the fact that events of speciation, Finally, the slope with which the level of diversity even if they implicate a process of inbreeding, do allow decreases with genetic distance from the centre would for the conservation of high levels of genomic diversity8. provide an indication of whether the character that drove the fixation was recessive, and was hence probably If we consider the various scenarios envisaged in the involved in a phenomenon of saeptation, co-dominant, or previous pages, however, the levels of diversity would dominant, and hence corresponded to adaptive evolution not be expected to be distributed evenly over the whole (including mechanisms of reinforcement). If such an genome. Indeed, the genomic regions surrounding the exercise was carried out for tens of thousands of markers loci having contributed to driving the speciation would distributed over the whole genome in hundreds of be expected to have reached fixation very rapidly, and unrelated individuals belonging to the same species, this hence to show very limited diversity. Furthermore, the could, I predict, provide a very informative picture of the rate of fixation would be very different if they successive steps of speciation in the evolutionary history corresponded to recessive or to dominant characters. of that species10. Indeed, if a recessive character leading to saeptation is being selected for, it will necessarily be fixed very

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 rapidly in the saeptated population, and one would thus expect a few centimorgans of the genomic region surrounding the recessive allele to become fixed with it, and hence to harbour very limited diversity, and this would be even more true for co-recessive traits. Conversely, whilst the allelic frequency of an 9 If the selective force driving the selection was a advantageous dominant character will rapidly increase to particularly nefarious pathogen, however, it may well be 70 or 80 % in a population, it will take a very long time that it would have disappeared with it’s host, and all that to reach complete fixation, i.e. to eliminate all the non- would be left would be an allelic form of a gene that was advantageous recessive alleles. Somewhat ironically, it is once used as a receptor for a now long vanished actually inbreeding that would allow the elimination of pathogen. the last ancestral, recessive and less advantageous alleles, 10 via a mechanism equivalent to the one described in In this regard, I would not be surprised if a locus section I-3. Consequently, during all that time before having driven saeptation in the ancestors of the complete fixation of the dominant allele, there will be laboratory rat, Rattus Norvegicus, was one day found to many chances for crossing-overs to occur around the lie near the MHC because the rat MHC has been found to gene coding for the advantageous dominant trait, and the have a much more restricted diversity of MHC haplotypes than those found in Mouse or Human. Alternatively, it may be that the ancestors of the rat 8 The model of Transilience developed by Templeton population have gone through one or several tight [44] addresses similar issues from the point of view of bottlenecks, resulting in limited diversity of sequences population genetics. through the whole genome.

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V) The existence of species can only be transitory because it corresponds to a metastable equilibrium. The field of Taxonomy was initiated by the swedish In this regard, the estimated number of 5000 extant zoologist, Carolus Linnaeus, who, in his book Systema mammal species represents only a tiny portion, and Naturae (first edition published in 1735, tenth and last in mammal species are particularly short lived, with an

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 1758), recorded some 9000 species of plants and estimated average lifetime of just one million years, animals. Today, this number has reached several whilst reptiles, and species of higher plants and trees can millions, and it is estimated that around ten millions last over 20 million years. All in all, it is pretty clear that species of plants and animals of more than one very few of the species that we can find on earth today millimetre inhabit our planet [47], and this number were there 20 million year ago. As already underlined in probably corresponds to less than 1% of the species that the introduction, the somewhat uncomfortable, but have existed since metazoan life started on earth 1.5 inescapable conclusion from this observation is that all billion years ago, with an estimated average lifetime of a the species that surround us, including our own, are species around 4 million years [48, 49]11. bound for extinction.

11 If we consider that sexually reproducing eukaryotes have existed for 1500 million years, and if the average lifetime of a species has been 4 million years since then, this amounts to an average number of approximately 400 steps of speciation separating the species of today from the first metazoan ancestors. If, along the way, every atoms on earth (ca. 1050). From this type of calculation, species had speciated into two descendants every 4 we can see that the struggle for existence highlighted by million years, this would give a number of species equal Darwin and Wallace for individuals must also apply to to 2400 which is so big that my desktop calculator refuses species, and that the destiny of most species is either to to calculate it, but which I make out to be something near disappear, or sometimes to yield one, and seldom more 10120, which is a number vastly superior to the number of descendants.

28/09/10 24 The theory developed in the previous pages can very high probability of ultimate extinction, whilst the actually lead us to suggest an explanation for this latter would lead to an increased probability of formation observed inherent tendency of species to disappear over of saeptating group(s) within the population, that will time. Indeed, we have seen that the mutation load in a ultimately cause the elimination of the ancestral group by population is inversely related to the degree of one or more descendant new species. The outbreeding inbreeding in this population, and the existence of strategy, however, is probably the one that takes place species thus appears to rely on a fragile, metastable most frequently in natural populations because it brings equilibrium, which I find very appropriate to represent in on much more immediate advantages. Darwin and the context of the Yin Yang symbol to evoke the balance Wallace’s theory of evolution, which is concerned with between degrees of inbreeding and outbreeding (Figure the acquisition of new adaptive traits, is in fact based on 3). considering this type of strategy. And it is indeed by relying on the flexibility and variability of the genome On the one hand, increased inbreeding will have the taking place in parallel in the numerous individuals of a various advantages listed in section I : reducing the cost large population that one can hope to see surface the very of sex, favouring the expression of recessive phenotypes, rare events that will correspond to new adaptive decreasing the mutation load, and promoting functions. Since such new traits will, most of the time, be collaborative behaviours by population fragmentation. expressed in a dominant fashion, they will thus rapidly But, as we have seen, this increased inbreeding will at spread to the whole population. the same time favour the appearance of saeptated groups, On the other hand, there are many instances where it is for whom the way of existence will equate to the advantageous to get rid of a character, and the elimination of the ancestral stock, and hence the susceptibility to pathogens seems to be particularly disappearance of the initial species. relevant here. But, as discussed at length in the previous pages, the loss of a function usually corresponds to a On the other hand, extensive outbreeding will bring recessive trait, and the expression of recessive traits hybrid vigour, and delay the appearance of reduced necessarily calls for some degree of inbreeding. And, fertility due to the accumulation of recessive deleterious although it is not endowed with the same immediately mutations. This type of phenomenon may be particularly obvious advantage as outbreeding, inbreeding does prominent for very successful species that end up reduces the cost of sex, and will keep the mutation load effectively panmictic rather than being fractionated into in check, although at the cost of reduced fertility. Given smaller subpopulations. The evolution of individuals the less tangible and more delayed nature of those within such population would then favour the strongest, advantages, and despite the reasoning developed in the longest lived, largest individuals. In this regard, van previous pages, I have actually found it very difficult to Valen underlined that, for mammals, “Occasionally, a convince myself that the prolonged existence of species small mammal becomes a large one, but a large mammal could rely on such a balance between outbreeding and never becomes a small one” [50] 12. Regarding the fossil inbreeding. record on which van Valen based most of his work, it may in fact be worth to consider the possibility that it’s But the degree of inbreeding necessary to keep composition may be biased towards species that, having mutation loads in check is probably much less than that adopted a panmictic strategy, would see the size of the required to promote speciation, and if we consider the

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 populations swell very rapidly to very large numbers, but very divergent outcomes of the two strategies, and the would at the same time rather rapidly reach a point of no timescales involved in evolutionary processes, we return because, after only of few dozens of generations, rapidly reach the conclusion that, in the long run, there is the accumulation of recessive mutations would not really a choice between the two strategies in the subsequently prevent any chance of any significant struggle for survival because extensive outbreeding will degree of inbreeding, and hence any possibility of a fresh inescapably lead to extinction. For example, this type of start via an event of speciation. And, as we have seen, situation may well have applied to the Multituberculates, such populations would inescapably at some stage fall which were very common mammals during the victims of their own success because they would be paleocene, but underwent complete extinction during the particularly prone to crises triggered by increased levels Eocene [51], probably because of the competition of the of selective pressures by outside factors such as newly arisen rodent competitors. If there are 10 million pathogens, predators, competitors, or a shift of the species on our planet, and the average lifetime of a climatic conditions. species is 4 million years, then the turnover rate should Hence, one major difference between the outbreeding be under three species per year. This may appear as a and inbreeding strategies is that the former leads to a clear underestimate, especially in our modern era where ecological changes due to human activities have

12 provoked the disappearance of thousands of species It is important to note that these rules do not seem to every year, but we should bear in mind that the apply to island mammals that are larger than rabbit size, extinction rates measured by paleontologists are those which tend to become smaller there, and mammals that which correspond to the disappearance of organisms are smaller than rabbit to become larger, which van based on the anatomical features detectable in fossils, Valen called the island rule.

28/09/10 25 and that species differentiated by colours, timing of life and cross, and will even attack one another. On one of cycle or breeding habits would not be registered. the Faroe Islands, not more than half a mile in diameter, Similarly, events of speciation corresponding to the loss the half-wild native black sheep do not readily mix with of one or a few recessive traits would almost certainly imported white sheep. In the Forest of Dean, and in the not de detected by the fossil record. Based on the New Forest, the dark and pale coloured herds of fallow arguments raised above, I perceive that most events of deer have never been known to mingle; and even the extinction identified by paleontologists probably curious Ancon sheep of quite modern origin have been correspond to those of panmictic species having observed to keep together, separating themselves from succumbed to increased selective pressures which the rest of the flock when put into enclosures with other initiated a process of irreversible decimation because of sheep. The same rule applies to birds, for Darwin was high inbreeding depression resulting from important informed by the Rev. W.D. Fox that his flocks of white mutation loads. From the above arguments, I conclude and Chinese geese kept distinct. that, even if inbreeding is not immediately advantageous, This constant preference of animals for their like, even it is an absolute requirement, an unavoidable price to in the case of slightly different varieties of the same pay, for long term survival of the descendants. This species, is evidently a fact of great importance in probably provides the ultimate example of group level considering the origin of species by natural selection, selection because species that fall for the short sighted since it shows us that, so soon as a slight differentiation advantage of extensive outbreeding will relatively of form or colour has been effected, isolation will at once rapidly have to face the cost of unmanageable mutation arise by the selective association of the animals loads, leading to unavoidable extinction. themselves; and thus the great stumbling-block of "the swamping effects of intercrossing," which has been so If we now turn to the world around us, we will see that prominently brought forward by many naturalists, will natural populations are, in fact, almost never panmictic. be completely obviated. First, the world is so vast that most species are necessarily fragmented into myriads of small groups, Such types of preference for closely related individuals with every event of colonisation providing an may not need to be based on purely genetic factors, but opportunity for episodes of increased inbreeding, could be culturally inherited, i.e. transmitted as memes resulting in a reduction of the mutation load. And there is [5], as has been documented many times with the also a natural tendency for individuals to associate with phenomenon of imprinting in birds raised in nests of kin, as Wallace himself underlined in the following different species. In addition, there is probably also paragraph taken from his book, ‘Darwinism’, in Chapter simply a natural tendency of individuals with similar VII’s section entitled ‘The Isolation of Varieties by phenotypes to breed more willingly and effectively with Selective Association’, (1889), which I do not resist the one another. pleasure of sharing with you: The concept that social structures and altruism are more likely to arise between genetically related But there is also a very powerful cause of isolation in individuals was initially developed by Hamilton [24, 25], the mental nature—the likes and dislikes—of animals; and this was later coined as the green beard altruism and to this is probably due the fact of the comparative effect by Richard Dawkins [5], to describe a hypothetical rarity of hybrids in a state of nature. The differently gene that would result in both a detectible trait and in

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 coloured herds of cattle in the Falkland Islands, each of altruistic behaviour among those bearing it. The which keeps separate, have been already mentioned; and occurrence of such a gene seems, however, rather it may be added, that the mouse-coloured variety seem to unlikely, and there are, indeed, very few reported have already developed a physiological peculiarity in occurrences of such possible green beard genes. breeding a month earlier than the others. Similar facts Moreover, the green beard hypothesis posits that the occur, however, among our domestic animals and are green beard would be a dominant character, i.e. a gain or well known to breeders. Professor Low, one of the a change of function, which, as underlined repeatedly in greatest authorities on our domesticated animals, says: the previous pages, is far less likely to arise through "The female of the dog, when not under restraint, makes mutations than a loss of function. selection of her mate, the mastiff selecting the mastiff, the terrier the terrier, and so on." And again: "The Merino More recently, however, mathematical modelling of sheep and Heath sheep of Scotland, if two flocks are beard chromodynamics yielded the conclusion that the mixed together, each will breed with its own variety." most stable arrangement for the maintenance of altruism Mr. Darwin has collected many facts illustrating this was for a situation where beard colours are polymorphic, point. One of the chief pigeon-fanciers in England and the genes for altruism only loosely coupled to those informed him that, if free to choose, each breed would for beard colours [52]. In other words, populations are prefer pairing with its own kind. Among the wild horses most likely to get organised into groups of individuals in Paraguay those of the same colour and size associate that behave altruistically towards one another if the together; while in Circassia there are three races of polymorphism of characters in the global population horses which have received special names, and which, allows individuals to recognise those that are most likely when living a free life, almost always refuse to mingle to be genetically related to themselves, i.e. the ones that

28/09/10 26 look like them, and the social genes do not have to be the V) Many classical examples of speciation appear to same as those used to evaluate kinship. In French, we fit the model proposed. have a proverb that says ‘Ce qui se ressemble For a scientist, one of the main problems in trying to s’assemble’, and the existence of races and varieties in understand the phenomenon of speciation is that it is the natural world vouches for the spontaneous basically impossible to perform experiments that will occurrence of structuration of natural populations which lead to bona fide speciation, i.e. complete reproductive can only be the result of some preferential association, isolation between two groups of individuals. The first and reproduction, between individuals that are more reason has been dubbed a ‘methodological contradiction’ closely related to one another than to the rest of the by Lewontin in 1974. Indeed, studying the genetics of population. The recent finding that, even in the fungus speciation involves experiments that cannot be done, i.e. Neurospora, some degree of reproductive isolation could cross species that are, by definition infertile with one be observed between stocks that had been grown for another. relatively short periods in different selective The second reason is one of the time scale, and/or of environments [53] indicates that a tendency for the size of the samples required. Indeed, because the preferential mating with individuals bearing similar mutations that lead to evolution and/or speciation occur phenotypes can occur even in microscopic organisms. purely by chance, they will only ever occur very rarely. In the previous sections of this essay, I have argued that For species that live exclusively on land, most niches speciation is promoted by inbreeding, i.e. by the small would naturally have patchy distributions, providing an size of breeding groups. But the probability of a new automatic enforcement of a fragmentation of mutation occurring in the very few individuals of that populations. But for species that live in the sea, or that breeding group is consequently infinitely small, and if can take to the air such as birds or insects, there will be one started from just one such breeding group, one no enforced limitation to taking advantage of the short would probably have to wait for thousands of term benefits of extensive outbreeding. In this regard, it generations for saeptation to occur. This is probably is actually remarkable to note that many species of birds, similar to what happens on isolated islands, where the fish, or marine mammals not only show strong time scales estimated to reach speciation are of the order preference for kin characters, but also come back to of tens of thousands of years (and no one would ever get breed to the very same place where they were born. I funding for an experiment on this time scale ;-). Of note, contend that these tendencies, which all must promote a in the Park Grass Experiment performed in East Anglia, significant degree of inbreeding, are the result of group adjacent plots of meadow have been continuously selection, with the groups or species adopting more subjected to different fertilizer treatments since 1858, outbreeding strategies succumbing rapidly to and some signs of reproductive isolation, with unmanageable mutation loads. Individual examples of reinforcement via different flowering times, have been various mechanisms promoting inbreeding will be identified between populations derived from one type of developed in the next section. grass [57]. I could, however, find no reported data regarding the reciprocity of this reproductive isolation, or The picture we come to at the end of this section is about the character which may have been selected for indeed one of a Yin Yang equilibrium between (and hence even less about its dominant or recessive outbreeding and inbreeding, in line with the notion of nature).

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 optimal outbreeding proposed by Bateson [54], whereby outbreeding is necessary for the acquisition of new Another approach to test the validity of a model of characters favoured by the parallel evolution of the many speciation is to make predictions regarding the type of individuals in whole populations, and inbreeding is results that could be expected from the model proposed, necessary to eliminate not only the deleterious recessive and then to check whether those predictions hold up mutations, but also to lose certain functions that have when the genetic source of reproductive isolation is become undesirable, and in particular the susceptibility dissected between closely related species. Many such to pathogens. Like many things in biology, including life predictions can thus be made from the model(s) itself [55], the existence of species has all the developed here, with the main ones being that i) When characteristics of a metastable equilibrium because speciation occurs, i.e. when a new group gets departing from it will promote a further departure from reproductively isolated from an ancestral population, this the equilibrium, either of outbreeding, destined for should be due to one or more recessive advantageous extinction, or inbreeding, which will favour speciation. mutation(s) occurring in the new group. ii) Genera that The observation of the constant rates of extinction within undergo a lot of speciation should be those that carry the genera reported by van Valen [21] does find an lowest mutation loads, correlating to lifestyles favouring explanation in this model because the occurrence of inbreeding such as frequent self fertilisation, or very events of destabilisation of the equilibrium would fragmented populations. correspond to presumably rare stochastic events, as was concluded by a recent study [56].

28/09/10 27 In the following paragraphs, we will look at several vibrant colours which characterised the males of the extensively documented cases of speciation that have various types of cichlids one hundred years ago been reported in the literature, and find that most of those disappeared progressively because the members of the examples do seem to fit the model proposed13. various ‘species’ hybridised with one another, to result in a much more homogenous and duller population [59]. Fish The first point to make from this observation is that, once The first ancestral vertebrate was a fish that lived in the again, it underlines the difficulty of defining species, ocean over 500 million years ago, and today more than since it shows that effectively good species can later on 30,000 species of fish occupy our planet’s waters, having become varieties when their living conditions change, adapted to the many diverse habitats found in oceans, even in the wild. From the point of view of the ideas seas, rivers, lakes… Since all these different species of developed in this essay, the observation that fish occupy niches of very different sizes and hybridisation resulted in the disappearance of the vibrant architectures, one would not expect the same colours is very reminiscent of the colours of Darwin’s inbreeding/outbreeding strategies to have been selected pigeons, and does thus strongly suggest that the in all fish, for example those breeding in the open ocean expression of many of these vibrant colours corresponds and to those living in lakes or in small streams. to recessive mutations, and that the expression of these Among fishes, the most often cited and discussed case must therefore require inbreeding. Conversely, another of speciation is that of the haplochromine cichlids found bright phenotype know as orange blotch is a dominant in the African lakes, which are hosts to hundreds of trait expressed only in females, to whom it brings a closely related species. Cihlids actually represent a very selective advantage [60]. This phenotype is encountered large family of over 3000 species which are widely in certain individuals of several species of the lakes distributed over the lakes and rivers of Africa and South Malawi and Victoria, but has not led to speciation. Thus, America (some of their best know representatives are the as predicted by the model, bright colours associated to Tilapia, which are used extensively for aquaculture, and recessive mutations lead to reproductively isolated the Angelfish, of Amazonian origin, which is commonly groups, whilst those associated to dominant mutations do found in domestic tropical aquaria). For many years, not. speciation of cichlids has been the subject of various heated controversies. The first one over whether the We will now turn to the reason why cichlids may have speciation see in African lakes really occurred in such a tendency to undergo explosive speciation, and I sympatry or allopatry, and a very strong argument for the contend that an explanation can be found in another fact that sympatric speciation was possible for cichlids recent study, based on Pelvicachromis taeniatus, a river- recently came from work on cichlids found in a very inhabiting African cichlid. In this report, Thünken and small crater lake of central America [58]. collaborators look at the mating preferences of this Another contentious issue has been whether some of cichlid fish, and find a strong preference for kin over the sympatric populations found in the African lakes non-kin, and, importantly, no sign of inbreeding represent varieties or ‘good species’. This latter question depression in the offspring resulting from sib mating is particular relevant for the diverse groups of Lake [61]. These observations strongly suggest that, in Victoria which were initially behaving as completely cichlids, there can be an inherent tendency to prefer isolated groups, because of assortative mating mostly mating with very closely related individuals. In rivers,

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 based on colour patterns. In recent years, as a result of where the context is presumably easily disruptive for human activity, the waters of the lake have become population structures, this preference for kin may be progressively more and more turbid, and the various important for the maintenance of low mutation loads, and for the expression of recessive phenotypes. When 13 cichlids with extensive inbreeding habits colonise lakes, In this regard, my relative naivety on the subject of however, the disrupting of groups of closely related speciation, to which I have already alluded to in the animals would no longer occur, and this could promote foreword of this essay, has proven to be a great frequent saeptation and result in the explosive speciation advantage. Indeed, it not only contributed to my capacity that is witnessed in African lakes. The existence of those to have ideas that seem to diverge quite significantly species will, however, be particularly fragile, because of from the currently accepted dogmas, but once I had the segmentation of the whole population into a myriad formally developed these ideas, it allowed me to gather of very small groups, that will be reproductively isolated data which already existed in the literature, but of which from one another, and will thus not benefit from sharing I was not aware, to test the validity of the model. In this their gene pool with a very large number of individuals. respect, I think it is worth underlining that my writing of And the high probability of saeptation of some subgroup all the previous sections preceded the writing of this will, in addition, represent another permanent threat for section, which coincided with my acquisition of the the occupation of the niche. The situation of cichlids thus knowledge about the precise details corresponding to the represents an inherently unstable situation, with an various models of speciation. I consider this to be a very excessive tendency for speciation. Ultimately, a more significant advantage because the model therefore has stable equilibrium would presumably be reached if some the added strength of having proven to be predictive rather than being adapted to explain the evidence.

28/09/10 28 sub-population lost its inherent preference for kin, and preferentially with individuals with which they share started adopting a more outbreeding strategy. some genetic similarity, but not too much, which is Although visual clues clearly play a pivotal role in the entirely compatible with the idea of a balance between preferences of cichlids for kin, another important inbreeding and outbreeding. component could possibly involve the MHC since, in Another fascinating observation made on sticklebacks other fish genera, MHC discrimination has been shown is the phenomenon of parallel speciation. Indeed, when to be involved in inbreeding avoidance. In this regard, populations of sticklebacks colonise freshwater the case of salmon provides a particularly interesting environments, and lakes in particular, they have a strong example of equilibrium whereby the MHC is used both tendency to evolve into adapted forms that lack certain by the mating adults for promoting MHC diversity (but features that characterise the anadromous (sea dwelling) not for inbreeding avoidance) [62], but conversely, sticklebacks: fertilisation is apparently favoured when eggs and sperm - In shallow lakes, one finds mostly a form called share MHC similarity [63]. Since, under normal benthic, which is larger, with smaller eyes, and feeds conditions, salmon copulation is usually polyandrous, mostly on invertebrates found on the lake’s bed. The and thus provides the grounds for sperm competition benthic form has a great reduction in the number of and/or sperm selection by the ovum, the choice of mates armour plates and of pelvic structures. would seem to play a relatively minor role, especially - In deep lakes with steep sides, the favoured since, as alluded to earlier, the tendency of salmon to adaptation tends to be a form called limnetic, which return to the very same place where they were born must corresponds to a smaller fish which feeds mostly on further enhance their tendency for inbreeding. In the plankton at the lake’s surface, and is, overall, less study by Landry et al., however, a relatively small different to the ancestral marine fish. number of males (41) and females (35) were placed in an In at least 6 lakes of British Columbia, Canada, arm of the same river that is not usually occupied by evolution has repeatedly driven the apparition of both salmon because of impassable waterfalls. Under such benthic and limnetic forms from the same ancestral conditions of unfamiliar grounds and low populations stocks. Those benthic and limnetic forms, although numbers, the results obtained by typing offspring for the capable of producing perfectly fertile offspring when MHC class II β chain suggested that matings had given no choice of partner, cohabit in those lakes in occurred to favour offspring that was heterozygous. In apparent complete reproductive isolation from one arctic charr, which is closely related to salmon, Olsen et another. For those various sympatric pairs, size and al. found that ancestry, whereby sibs were preferred to colour were shown to be the two main phenotypes that non-sibs, had a more important influence than MHC contribute most to the reproductive isolation [69]. A truly preference [64]. The fact that these two latter studies remarkable finding was the observation that, for all these looked at the MHC class II locus whilst the study of populations, benthic individuals from one lake mated Yeates et al. looked at MHC class I may be of some preferentially with benthic partners from other lakes than importance because, in teleost fish, the regions for class I with limnetic ones from any lake and the converse was and class II are not linked [65], and cues conveyed by true for limnetic fish [70]. As underlined by the authors, one class of MHC molecules could have different the observation that, under similar conditions, evolution consequences to those of the other class, which may have can lead to the parallel selection of similar sympatric contributed to the apparent discrepancy of some of those reproductively isolated populations is a very strong

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 results. argument in favour of the idea that natural selection is involved in the speciation process. This was in fact Sticklebacks are another type of fish which are found further supported by their observation that reproductive both in the sea and in fresh waters and have been very isolation between benthic and limnetic individuals useful for the study of speciation. Sticklebacks, which seemed even more pronounced between fish from the are also known as tiddlers in English, are very efficient same lakes than between fish obtained from different colonisers, and are widespread in the northern lakes, despite being more closely related to fish from the hemisphere, over much of Europe, Asia, and America. same lake. It thus suggested that reproductive barriers Regarding the role of the MHC in mating preference, an could be selectively raised against the population that intriguing observation is that sticklebacks appear to represented the most direct threat, i.e. the fish of the favour mating with individuals of intermediate MHC same lake, but of the other morph. divergence, to yield offspring with an optimal number of Further work, orchestrated by the group of David 5 to 6 MHC class II β alleles [66], which they apparently Kingsley, has led to the dissection of the genetic achieve by smelling the peptides that can bind to MHC mechanisms responsible for the loss of armour plates or molecules [67]. In another study, however, Frommen and of pelvic structures which are both particularly Bakker found some signs of inbreeding avoidance in prominent in benthic sticklebacks. As predicted by the groups of fish raised separately, but with no data on the model proposed in this essay, both phenotypes were correlation to MHC similarity [68]. From the point of found to be due to mostly recessive mutations. The loss view of the ideas developed in this essay, the data from of armour plates was mapped to the ectodysplasin gene ( salmon and sticklebacks contribute to the drawing of a EDA) which, in mammals, is known to be involved in picture whereby clues from the MHC allow fish to mate many ectoderm features such as teeth and hair [71].

28/09/10 29 Remarkably, the same allele of the EDA gene, which phenotype, which would increase the delay with which carries just four amino acid differences compared to that the genomic regions carrying the mutation would found in fully plated fish, was identified in all the low become fixed, and thus provide plenty of opportunities plated morphs obtained from Europe, and from both the for crossing-overs to occur in the close vicinity of that American coasts. That same allele was also identified in region. fully plated fish caught in river estuaries, albeit at low frequencies (3.8% in California and 0.2% in British Altogether, the picture that shapes itself regarding Columbia). Another allele was, however, found in speciation in sticklebacks adapted to lake environments Japanese stocks, which shows no changes from the wild is one where either hidden recessive phenotypes, or type in the protein coding sequence, but falls in the same relatively probable inactivating mutations initially result complementation group as the other low-plate in recessive advantageous phenotypes, promoting phenotypes. These results suggest that the allele successive steps of saeptation from the ancestral marine responsible for plate loss in sticklebacks has been around fish. Subsequently, once separate groups have been for several million years, and has spread widely over the formed, reinforcement based on sexual preferences will northern hemisphere, probably because it is associated to then follow, driven either by the ancestral stock or by the a very significant advantage in freshwater populations, other morph, based on a variety of phenotypes, among where it would thus get amplified, and then fed back into which size and colour are particularly prominent. the marine population by episodes of hybridisation. Because it is essentially recessive, this allele can remain So far, in this section, we have only considered fish ‘hidden’ at low frequency in the marine populations. species that are naturally structured and/or have been When marine stocks colonise freshwater niches, recognised as prone to undergo speciation (both factors however, this must favour some degree of inbreeding, actually going hand in hand if we accept the proposed which would rapidly reveal the recessive phenotype, and model). In the oceans there are, however, many other the selective advantage would then rapidly increase the types of fish populations that are extremely numerous, allelic frequency in the isolated population. In conditions and hence probably much more prone to panmictic where the threat of the fully-plated allele persists, this reproductive strategies. Those that spring to mind are, for will provide the grounds for selection of reinforcement example, mackerels, sardines, anchovies or cods, and for via mechanisms such as reproductive isolation, which all of those, great fluctuations of effectives have been could ultimately result in proper speciation. witnessed over the years, with recovery rates that often The loss of pelvic structures was also very recently prove difficult to predict. This is particularly true for the shown to be due to recessive mutations corresponding to cod populations, which are proving very slow to recover deletions in the promoter regions of the Pitx1 gene [72]. from the overfishing that has taken place over the past Remarkably, characterisation of the promoter regions of decades. In line with the model proposed in this essay, I this gene in nine different populations of benthic contend that, for fish populations that are sufficiently sticklebacks revealed that the same 488-bp segment was numerous to adopt a panmictic strategy, the variations in missing in all nine populations, but this was due to nine numbers, and in particular their episodic slow recovery different events of deletion. This observation thus after population shrinkage, could partly be due to testifies that the advantageous phenotype of losing pelvic reduced fertility caused by high mutation loads in the structures arose repeatedly and independently in all those context of increased inbreeding coefficients caused by

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 completely separate benthic populations as a result of population shrinkage. selective pressures, and, contrarily to the previous example, was not ‘hidden’ as a recessive trait in the To conclude about fish, the currently available data ancestral marine population, which would only surface suggest that, in species that tend to have a certain under conditions of inbreeding. The other point that can structure imposed by the niche they occupy and/or their be made from this observation is that, under the right breeding habits, mechanisms exist that would ensure a conditions of selection, recessive mutations due to loss of balance between inbreeding and outbreeding by existing genetics materials are sufficiently common that favouring mating between individuals of relative they can be repeatedly obtained in completely relatedness. When circumstances change, however, such independent populations. Another remarkable as when cichlids or sticklebacks find themselves in the observation contained in that article is that, as predicted more stable and secluded environment of a lake rather at the end of section IV, there is a considerable reduction than in streams or the ocean, this will tilt the balance in the heterogeneity of sequences focused on the region towards inbreeding, and favour speciation. surrounding the Pitx1 gene [72]. Amazingly, this reduction only spreads over a few kilobases, which Birds suggests that events of DNA recombination such as For birds, the capacity to take to the air potentially crossing-overs must occur very frequently over the opens an almost limitless capacity for dissemination. region carrying this gene. As discussed in section IV, the Many bird species are, however, rather sedentary, with a tightness of the region of reduced polymorphism may strong tendency for territoriality. And for those that are actually be related to the fact that a sizeable proportion migratory, similarly to fish, there is a very strong of the Pitx1 mutations have a completely recessive

28/09/10 30 tendency to return to the very place of their birth when beak shape (incidentally, the shape of the beak has by they reach sexual maturity. itself a strong influence on the song). The main factors Contrarily to fish, however, there is no clear sign that that control the shape of the beak have actually been the MHC plays a strong role in regulating the relatedness indentified as bmp4 (depth and width ) [74] and of mating partners, probably because the sense of smell Calmodulin (length) [75]. Both factors act independently is less developed in birds than in fish. Rather, visual and from one another, and in a dose-dependent manner. The auditory clues are used extensively in the establishment various beak phenotypes are thus expected to behave as of the usually monogamous breeding pairs. Remarkably, co-dominant traits (or rather co-recessive, since hybrids rather than being innate, sexual preferences of birds are would thus express intermediate, less suitable actually mostly cultural, i.e. mainly acquired via a phenotypes). mechanism called imprinting, which takes place during Since some hybridisation (of the order of a few %) the first few weeks of life. One must, however, underline between certain species can still occur [76], a dogmatic that there must also be some level of innate capacity of evolutionist could argue that those populations thus do certain birds to recognise kin. Otherwise, how would the not represent true species. For the purpose of the ideas cuckoo ever recognise it’s mate? During the imprinting developed in this essay, Darwin’s finches simply provide period, birds learn to identify various characters such as a very telling example of a population of individuals the song of their parents, as well as the size, shape and founded by a very limited effective. In the restrained colours of their parents’ or siblings’ anatomical features context of those small islands, inbreeding coefficient such as beaks or plumage. Imprinting has been were thus necessarily increased, and, given the natural demonstrated in too many bird species to cite them all propensity of birds to prefer mating with close kin, and here, with varying degrees of importance put on song or the co-recessive nature of the traits selected, this anatomical features depending on each species. The most situation has led to one of the most impressive examples picturesque and best know example is certainly that of of adaptive radiation documented to date. the experiments performed with geese by Konrad Lorenz where he showed that the goslings became imprinted on Mammals: him (or more precisely on his gumboots) during the first Contrarily to fish and birds, most mammals are few hours after their hatching. When it comes to restricted in their dispersion (the technical term for this is choosing a mate, those preferences would hence promote limited vagility), and most populations of mammals are pairing between closely related individuals. Working thus naturally fragmented, and this is particularly true for with Japanese quails, Bateson actually demonstrated that those that live in relatively small groups, such as horses cousins were the preferred partners, i.e. individuals that or certain primates, or are active colonisers, such as differed a little bit from the parental picture, but not too murine rodents (rats and mice)14. When the natural much [73]. Based on his observations, Bateson proposed tendency of a species is for a small number of the notion of ‘optimal outbreeding’[54], which could not individuals to find themselves repeatedly isolated into possibly be more in line with the ideas put forward in separate colonies, thus imposing high inbreeding this essay. coefficients, it is expected that the natural instincts Since one could not possibly evoke the subject of should evolve to compensate for this, and thus favour speciation in birds without mentioning the most outbreeding whenever possible rather than further emblematic case of Darwin’s finches, I will briefly inbreeding.

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 discuss those as a final example. Those famous finches Such behaviours have indeed been documented in were collected by Darwin (or more precisely shot and many mammalian species, and in particular in the house preserved by his servant, Syms Covington) on the mouse, Mus Musculus domesticus. For many years, Galapagos islands during the second voyage of the experimental evidence has been accumulated showing Beagle, and only identified later by the ornithologist that there was indeed inbreeding avoidance between John Gould as a new group of twelve separate species of mice from different inbred strains, and documented that finches which seemed most related to ground finches the MHC was playing a pivotal role in this phenomenon. found on the south American continent. Today, Darwin’s More recently, however, the group of Jane Hurst used finches are classified into fourteen different species that wild mice rather than inbred strains to document the have different distributions on the different islands of the mating behaviours of mice, and identified that major archipelago, and for which the most telling anatomical difference lies in the size and shape of the beaks, which 14 are variously adapted to feed on different nutriments The fact that it has been possible to generate (different size of seeds, different parts of cactuses, or consanguineous lines of rats and mice has proven various other sources such as insects or larvae). extremely useful for scientific research. For other species Molecular characterisation of those different species has such as rabbits, hamsters or guinea pigs, this has, led to the conclusion that all those species derive from a however proven much more difficult. I contend that this common ancestral stock which probably comprised at could in part be explained by the natural tendency of least 30 founders ( C&O, p 403). There is clear muridaes to colonise new environments, which must reproductive isolation between the various species, with have kept their mutation loads very low, and also shaped imprinting documented to occur both on songs and on their genomes to cope with repeated episodes of extreme inbreeding.

28/09/10 31 urinary proteins ( MUPs) had a much more potent offspring of a colony. One of them is the African wild influence on kin recognition, and inbreeding avoidance, dog, which lives in pack of 20 to 40 animals, and than did the MHC [77]. The discrepancy between those inbreeding avoidance is obtained by males and non- results and those obtained previously by other groups reproducing females emigrating away from the finds an explanation with the fact that the process of population. Another one is the eusocial naked mole rat, deriving inbred mice has yielded strains with very which is found in east Africa and is actually more closely limited inter-strain variability of the MUPs [78]. related to porcupines than to rats. Those live exclusively Furthermore, in an extremely recent paper, the group of underground, in colonies of 50-100 individuals where all Jane Hurst actually characterises Darcin, an invariant the offspring descends from one single ‘queen’. urinary protein found in the urine of male mice, which Although inbreeding coefficients have been found to be behaves as a pheromone by inducing contact-dependent extremely high in those animals, this must be a imprinting of females to prefer the males harbouring the consequence of their lifestyle rather than by choice since other smells found in that urine [79].The observation that outbreeding was found to be preferred when available diverse MUP complexes undergo parallel evolution in [86]. different species suggests that polymorphic MUPs, as well as other polymorphic factors [80], may play an Inbreeding is, however, not avoided to the same degree important role in regulating the mating behaviour in in all mammal species, and there are also numerous many species [81], which may call for revisiting some of examples of kin preference in mammals, which are often the results obtained regarding the pivotal role of the the result of imprinting, in other words a cultural rather MHC in regulating the degree of inbreeding between than a genetic heritage. On the whole, one finds that individuals in vertebrate species, including fish. mammals in which inbreeding avoidance is the most The precise mechanism(s) driving inbreeding prominent are those for which their natural lifestyle avoidance is, however, of little relevance to the ideas would most often provoke the isolation of small groups. discussed here. Rather, we can find multiple arguments Yet, they should presumably be those carrying the that provide strong support for the ideas proposed here in smallest mutation loads. Hence, they should be the ones the study published by Bush et al. more than 30 years for which inbreeding is the less costly. For mammals as ago [82]. for fish and birds, the overall picture therefore seems to Firstly, they underline that the effective size of match a model of balance between inbreeding and mammal populations (which is inversely correlated to the outbreeding, in line with Bateson’s optimal outbreeding average inbreeding coefficient) appears to be inversely model [54] rather than outright and systematic correlated to the rate of speciation: Whilst speciation is inbreeding avoidance. One study carried out in wild very rapid in horses and primates, which have very American Pikas (which are related to rabbits and hares) structured populations, it is much slower in marsupials actually found that, much like Bateson’s Japanese quails, and carnivores, which have much more diffusive the preferred partners were those of intermediate breeding strategies, and slowest in bats and whales, relatedness [87]. probably because of their high vagility. The various altruistic behaviours frequently witnessed in certain Insects colonies of bats is, however, often viewed as being due With more than one million species identified, the class to high levels of relatedness between the individuals of the insects is, by far, the most numerous one of the

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 comprising those colonies. One could thus envisage that whole kingdom of eukaryotic life, and basically the longevity of bat species may relate to the stability of comprises half of the metazoan species recorded to date. the equilibrium between outbreeding (due to their high Insects are thus clearly very prone to speciation. vagility) and inbreeding (due to the structure of their Although insect populations are often very large, they are colonies, promoted by the importance of cooperative also very frequently fragmented into very restricted and behaviours for their survival [83]). diverse niches, which often exist only transitorily, and Second, the rate of speciation is also shown to be which must thus be repeatedly colonised by a handful of strongly correlated to the rate of chromosomal evolution, individuals. To my knowledge, no behavioural and horses, primates and rodents are indeed genera inbreeding avoidance has ever been described in insects, where many instances of chromosomal rearrangements and it is only very recently that some level of have been documented between closely related species, outbreeding preference has been reported, in polyandrous which can sometimes produce hybrids that are either female field crickets, via a process of preferential sperm- infertile (as for the equine species) or of limited fertility, storage [88], and this despite similar success of mating as for the chromosomal species of alpine mice [84, 85]. with sibs or non-sibs [89]. Conversely, numerous Third, the authors also underline that the organisation instances have been documented whereby insects show of populations into clan or harems, where a single kin preference, based on a whole range of processes dominant male sires most of the females is another which include preferred mating protocols, acoustic and mechanism which reduces the effective size of visuals clues and pheromone detection. Repeated populations, and thus increases the average inbreeding episodes of colonisation, and the absence of inbreeding coefficient. There are a few notable exceptions where it avoidance must contribute to keeping the mutation loads is actually the female that gives rise to most of the down, and thus promote the phenomenon of speciation in

28/09/10 32 insects. As developed in addendum 2, the haplodiploid reproductive isolation arises as a succession of small mode of reproduction of insects such as the hymenoptera steps, each selected for under the threat of another (ants, bees, wasps … ) corresponds to a very effective population : either the ancestral population expressing a way of eliminating recessive mutations, and it is quite dominant but deleterious trait, or a newly arisen group remarkable that the hymenoptera represent more than which threatens the ancestral population by progressively 30% of all insect species. Another factor which may taking over the niche. contribute significantly to the tendency of insects to - Multiple studies, of which many come from the group undergo speciation is that the selective pressures due to of Mohamed Noor, underline the implication of predation are particularly significant for insects, and chromosomal rearrangements in the reproductive traits that can reduce detection by predators are quite isolation seen between closely related species of often recessive. drosophila. When they have been mapped, the genes for female preference and hybrid male sterility were found Among all insects, Drosophila has proven a to be associated with chromosomal rearrangements [38, particularly useful tool for many aspects of biology, and 91], and furthermore, such rearrangements are much particularly for genetics and the study of speciation. So more prominent between sympatric species than between much data has been published on speciation in allopatric ones ( [38] C&0, p309 ). The explanation most Drosophila that it would be unrealistic to attempt to commonly offered for these observations, in line with the summarise it here (there are more than 50 sections Dobzhanski-Muller model, is that the chromosomal discussing Drosophila in the book Speciation by Coyne rearrangements prevent recombinations between multiple and Orr (2004), many of them several pages long). I will genes having co-evolved. As proposed in section II 5c, if therefore restrict myself to outlining a few points that reproductive isolation evolves as a response to the threat seem to be most relevant to the ideas developed in this of hybridisation with a neighbouring distinct population, essay. chromosomal rearrangements could also have two Regarding genetic loads in fragmented populations, as additional effects contributing to the isolating early as 1964, Dobzhansky was underlining the phenotypes: first induce some level of infertility in observations made by several groups that “the heaviest hybrids, and second be endowed with an intrinsic genetic loads are found in common and ecologically phenotype, either by the inactivation of a gene leading to most versatile species of Drosophila, and the lightest an advantageous recessive phenotype, or by modifying ones in rare and specialized species and in marginal the genomic context of the genes surrounding the colonies of common ones” [90]. rearrangement. Reproductive isolation between different species of Drosophila relies mainly on two mechanisms: choosiness Altogether, the masses of data accumulated with of females for the males of their own species, and hybrid various species of drosophila seem to be in perfect sterility. agreement with the model proposed, whereby the flies’ - When crossings occur between different species, lifestyle, which involves repeated colonising of isolated mating preferences almost systematically disappear in F1 habitats by a few individuals, results in very fragmented females ( C&O, chapter 6), which testifies for the populations, with high inbreeding coefficients and recessive nature of those phenotypes. If we follow the consequently low mutation loads. This must increase the type of reasoning developed in the previous pages, this probability of both the appearance of advantageous

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 would suggest that such characters leading to recessive phenotypes, and fixation of chromosomal behavioural isolation must have arisen in the context of rearrangements. Whilst the resulting groups would not saeptation, which could have been either primary, or initially be strongly infertile with the ancestral secondary to the constitution of two populations. The population, hybridisation will be detrimental to the repeated observation that stronger assortative mating is fitness of the offspring, which would promote found between populations of flies that are in close reinforcement mostly in the more threatened, less contact in the wild (C&O, p 357-365) brings very strong numerous newly arisen group, thus explaining the support to the idea that reproductive isolation is a asymmetry of the isolation phenotypes often observed phenotype that is selected for, and not just the result of between drosophila populations. drift between populations that are not in contact with one another. Another fly species which is an old favourite for the - Regarding hybrid sterility, it follows Haldane’s rule study of speciation is the maggot fly, Rhagoletis since it is almost always the males that are sterile. A pomonella, which one finds in North America and which large body of evidence from various studies suggests that adapted very rapidly from its native hawthorn host to this sterility is often asymmetric (ie concerns the males cultivated apples after those were introduced in north obtained through only one of the two types of crossings), America in the 1800’s, and the first report of this and results from the accumulation of multiple small speciation can be traced back to Walsh in 1867, less than effects mapping to various genes rather than to the large 70 years after apples were introduced, and very soon effect of major genes ( see C&O, p299-319). This is in after Darwin’s publication of The Origin. Although some complete agreement with the scenarios proposed in gene flow can still occur, there is very significant section III and sketched in figure 2, whereby reproductive isolation between the two species, based on

28/09/10 33 a combination of factors which include the fact that the early steps of speciation [95]. In western Ecuador, larvae have different timings for their emergence from one finds Heliconius cydno alithea, which is a mimetic their diapause (i.e. the larval life), leading to adults butterfly which follows the models of other Heliconius having reduced overlapping periods for hybridisation, butterflies, H. sapho (white) and H. eleuchia (yellow). and also that Rhagoletis mate on or near the fruit of their Those two latter species produce toxic chemicals that host plant. The data on Rhagoletis fits the proposed protect them against predation. Within the population of model very well: Firstly, preferential responses to H. cydno alithea, depending on the region, one finds specific fruit odours are recessive since they have been white and yellow butterflies in various proportions, shown to disappear in F1 hybrids [92]. Second, multiple which correlate with the relative abundance of the loci related to diapause have actually been mapped to respective white and yellow models in that same region. regions of chromosomal rearrangements which have Whilst white and yellow H. cydno alithea are not been shown to have introgressed from an isolated reproductively isolated, Chamberlain and colleagues population of Mexican Rhagoletis. The overall picture is found that the yellow males showed a marked preference thus one where recessive odour-based fruit preference for yellow females, whilst white males were would drive saeptation, and chromosomal indiscriminate. Crosses between yellow and white rearrangements associated to different diapause butterflies also revealed that white is the dominant phenotypes would reinforce the isolation both by phenotype. Remarkably, male preference was found to favouring intra-group synchrony, and presumably also by segregate with the K locus coding for wing colour, which reducing inter-group fertility. may be explained by the fact that the same pigments dictating wing colour are also used as filtering pigments Chromosomal rearrangements are found in closely in insect eyes. related species, or sub-species in many other types of Thus, in butterflies as in cichlids, selection of insects, and the best documented example is probably in advantageous recessive colour patterns can lead to some the Australian wingless grasshoppers, which were degree of reproductive isolation, whilst dominant ones studied by White, and which led him to propose the do not. model of stasipatric speciation [93](C&O, p16), and more recently that of chains of chromosomal changes Flowering Plants [94], whereby sequential chromosomal rearrangements With close to 300.000 species recorded, flowering progressively reinforce the genetic isolation of a plants compete with arthropods for the second place for population from the ancestral one, in conjunction with the phylum with the most species [47, 96]. Among those, other mechanisms of reinforcement such as hybrid the rate of speciation appears to be particularly sterility [93]. prominent in plants capable of self-fertilisation. This is in part related to the phenomenon of speciation by If we now turn to butterflies, we can find two examples polyploidy, which is actually relatively rare, and occurs that underline the correlation between the recessivity of over just one or very few generations, and is thus not phenotypes and the phenomenon of speciation. The first really relevant to the mechanisms we are trying to dissect example is that of the peppered moth, Biston betularia, in this essay ( see C&O, chapter 9). As first proposed by which was first reported by J.W. Tutt in 1896, and has Baker in 1953, the higher number of species among since become an emblematic example of adaptive selfing plants is often interpreted as related to their

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 evolution. Originally, the populations of those moths higher capacity to colonise new environments (see[97]), were light colored (peppered), which provided very good and this does indeed fit the para- and/or allopatric camouflage against the barks of trees. During the scenarios proposed in section III. industrial revolution, however, many lichen died, and the An additional factor may, however, be that the capacity average color of tree trunks turned much darker because to self fertilise, which is the ultimate form of inbreeding, of soot deposits. This made the light colored peppered would be very effective at reducing the mutation load, moths much more conspicuous for their bird predators, which would, in turn, favour speciation. In a more recent and led to the selection of a darker phenotype, the black- report Heilbuth concluded that it was not so much the bodied moth, which initially represented less than 2 % of capacity to self-fertilise that increased speciation, but individuals, but raised to around 95% over the five dioecy (i.e. the complete separation of the population decades between the middle and the end of the 19th between males and females) that was associated with century. With the color of tree trunks progressively lower number of species [98], which is in complete returning to a more natural light color, the frequency of agreement with the observation that dioecious plants dark moths has since been decreasing slowly. The only comprise 6% of all flowering plants, among which rapidity of the initial selection process is explained by one finds Holy, Willow, Ash, Juniper and Gingko biloba the fact that the darker phenotype is due to a dominant (one of the longest lived species know to date, C&O, mutation. In fitting with the model, this did not, however, p425). To reach this conclusion, Heilbuth compared lead to any detectable process of reproductive isolation. multiple plant families for species richness among three Conversely, in another butterfly, a recent report types of plants : those capable of selfing, those where describes that a recessive phenotype is associated with selfing could be possible but is prevented via various the type of mating preference expected to correspond to self-incompatibility mechanisms, and dioecious plants,

28/09/10 34 and found comparatively low numbers of species only in Phylogenetic comparisons have established that the the latter. This puzzling observation can, however, find ancestral phenotype was the paler colour of M. lewisii, explanations in the light of the model proposed here. and the bright colour of M. cardinalis is actually a Indeed, the prediction from the model is that, under recessive phenotype due to a mutation in the YUP gene, conditions of excessive inbreeding, populations will which prevents carotenoid deposition in the petals [101]. undergo very frequent speciation, but the durability of By deriving near-isogenic lines of Mimulus for various these species will, consequently, be much reduced characters, Bradshaw and Schemske managed to because most new species will tend to eliminate their demonstrate that, although the two species have diverged immediate ancestor. The incapacity to self fertilise may by many other detectable traits that segregated diversely, thus reduce the rate of speciation, but would increase the the preference of either hummingbirds or bumblebees lifetime of the species, with a net result of equivalent was primarily controlled by this single locus. Hence, we numbers of species. Furthermore, the diversity of have here an example where a recessive mutation has led mechanisms used for self-incompatibility in various to what I consider a clear case of parapatric speciation by plant species suggests that those have been repeatedly provoking a switch to a different pollinator, which and independently selected for, probably because they presumably allowed M. cardinalis to colonise the lower represented a selective advantage in populations that had ranges and valleys where hummingbirds are found. an excessive tendency to undergo rampant speciation. In addition to underlining the correlation between the The last plant example I have chosen to discuss is that selfing capacity of plants and their propensity to colonise of oaks (Quercus), for their extraordinary capacity to remote grounds, Baker was also among the first to resist speciation, since complete reproductive isolation propose that sex could have evolved as a mean to reduce still has not been reached between many of the inbreeding [99]. Multiple arguments exist to suggest that approximately 400 ‘species’ of oaks recorded to date. dioecy is a much more efficient guard against inbreeding Those correspond to very different types which are than mechanisms of self incompatibility such as distributed over very spread and diverse habitats of the gametophyte incompatibility ( see [98]), and switches northern hemisphere, but many of those ‘species’, and between dieocious and selfing modes of reproduction particularly the group of white oaks which are most must also be much less likely than between self- prominent in north America, can still intercross and yield incompatible and -compatible ones, such as recently perfectly fit offspring, with clear signs of hybridisation described for the annual plant Capsella [100]. Given this, and gene flow having been documented in wild it is thus not surprising that dioecious species should be populations [102-104]. Whilst these observations have guarded against inbreeding via higher mutations loads, led to numerous and lengthy debates on the and thus have a much lower tendency towards speciation, appropriateness of such or such definition of species, and and thus be much less numerous than those with left evolution biologists puzzled for many years, the very complete or partial hermaphrodism. One should not, limited tendency of oaks to undergo speciation may also however, make the mistake of equating speciation with find an explanation in the model proposed here. Firstly, adaptive evolution. Evolution is related to the acquisition although oaks are monoecious, gametophytic self- of new characters, which is much favoured by the incompatibilty has been found in many types of oaks. exchange of genetic material among numerous Second, oaks have a very significant capacity to spread individuals in large and long established populations. both via pollen and via acorns that can be transported

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 Speciation, according to our model, is mainly due to the over rather short distances by animals such as squirrels, loss of some undesirable trait(s), which can only occur but also over much longer distances by floating down via inbreeding between a necessarily restricted number streams, or conceivably even across sea waters (Darwin of individuals, which will almost always result in some spends several pages of The Origin discussing the loss of diversity, and thus reduce further adaptability. resistance of various seeds to seawater). This capacity to diffuse must greatly favour hybridisation, and thus the To conclude on plants, we will turn towards a couple of spread of dominant advantageous traits. At the same examples of plants which are common favourites of time, oaks have a clear tendency to congregate in forests speciation specialists. that are comprised mostly of oaks, and this must surely The first case is that of two closely related species of provide the grounds for a certain degree of inbreeding, monkeyflowers, Mimulus lewisii and cardinalis. Those which must result in the mutation load remaining are found in the hills and mountains of California, with reasonably low. Another factor that must favour the the pink M. lewisii occupying higher altitudes, and the selection of very vigorous hybrids lies with the number bright red M. cardinalis occupying the valleys. Although of acorns that an oak produces. Indeed, during its very the ranges of the two species overlap between 1500 and long lifetime, a single oak produces hundreds of 2000 meters, and despite the fact that they are capable of thousands of acorns, which will give several thousands producing viable and fertile offspring in the greenhouse, of seedlings, and maybe a few dozen young trees, but of hybrids are almost never found in the wild, which is which only a handful (two, statistically) will go on to highly related to the fact that the red M. cardinalis is produce progeny themselves. This tremendous level of pollinated mostly by hummingbirds, whilst the paler M. selective pressure probably contributes significantly to lewisii is pollinated almost exclusively by bumble bees. preventing the survival of those suffering from any

28/09/10 35 significant degree of inbreeding depression. Altogether, I Today, the human population comprises well over 6 would surmise that the situation of oaks probably hovers billion people, and this number is predicted to reach 9 near the equilibrium between the yin of inbreeding and billion in about forty years, despite serious uncertainties the yang of outbreeding, with a mutation load about the capacity of our planet to sustainably feed that sufficiently low to prevent degeneration, but sufficient many people. Although it is universally admitted that we outcrossing to share dominant advantageous phenotypes, all belong to the same species, humans are split into and at the same time maintaining a mutation load many ethnies and races. If one looks at the situation in sufficiently high to prevent the degree of close places where those ethnies come into close contact with inbreeding that promotes the successive steps of one another, such as in big cities, one does, however, saeptation leading to speciation. witness a very significant level of intra-racial preferential pairing. Furthermore, offspring of interracial couples, White Sand lizards: an experiment in progress: whilst benefitting from high physical fitness, often suffer In the first paragraphs of this section, I underlined why from reduced social fitness because they find themselves it was so difficult to carry out experiments related to struggling to integrate into either of the groups that their speciation. The example I have chosen to conclude this parents came from. In this sense, to highlight once again section is one where nature may actually have provided the difficulty of defining species, if one adopted the same us with such an experiment, including the indispensible criteria as are often applied to animal or plant species in internal control. In White Sands, New Mexico, USA, the wild, one could conclude that speciation has already dunes of white gypsum formed less than 6000 years ago. started occurring in humans. In support of this rather In those dunes, one finds several types of lizards provocative stance, the most distinctive phenotypes to harbouring very light colours, which are each descended distinguish between ethnies are the colours of skin, hair from their darker relatives found in the nearby and eyes, which have progressively gone from dark in Chihuahuan desert. Amazingly, in three separate species, our African ancestors to the very pale skin, blond hair the group of Erica Rosenblum has recently mapped the and blue eyes seen in Northern European populations. cause of this albinism to different mutations of the very And it is completely fitting with the model that same gene : the melanocortin-1 receptor (which is, secondary mechanisms of isolation such as xenophobia incidentally, also associated to red hair in human). Even or racism, should often be asymmetrical, and strongest more remarkably, in one species, the mutation is on the side that expresses the recessive traits. For dominant, whereas it is recessive in another one. example, we know that all the “arian” traits that were the Although the mating preferences between white and basis of the selection criteria of the Nazi doctrine do not brown lizards have not yet been documented for those actually correspond to real improvements by a gain of a two species, those experiments are being scheduled ( E. new function, but all correspond to mutations causing Rosenblum, personal communication). The prediction losses of function in various pigment genes, which are all from the model is that, if there is asymmetry in the either recessive, or co-recessive. Another even darker mating preferences, those should be stronger in the aspect of the human practices matches certain points morphs with the recessive form (white or brown), and discussed in section IV: War is very similar to a most prominent in Aspidoscelis inormata, for whom the sympatric struggle, i.e. a conflict for the occupation of white phenotype is recessive, and the threat of breeding the niche between separate populations. In times of with the more numerous ancestral stock of brown lizards conflict, sexual violence and systematic rape have been

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 thus much more significant. used for centuries as a weapon of war (see http://www.unicef.org/sowc96pk/sexviol.htm ). Indeed, in the context of a sympatric struggle, the practice of VI ) And what about Homo sapiens ? systematic rape is a very effective strategy to neutralise “Dans un oeuf, y'a du blanc et du jaune. Eh bien quand the reproductive force of the opposing population, and on mélange, il n'y a que du jaune”. Coluche, Les imposes a burden that can last for many years by Vacances (1979) producing children that are often rejected by both camps. Thankfully, since 1998, the United Nations as decided to In the paragraph discussing mammals, I purposefully consider this abominable practice as genocide, and as a avoided the difficult subject of the situation of the human crime against humanity. race. As we will see in the following paragraphs, there The recent discovery of a few percents of Neanderthal are many aspects whereby what we know of past and sequences in the genome of Eurasian populations and not current structures of human populations, as well as in those of sub-Saharan African descent [105] is also in human instincts appears to fit the model, if only too well perfect agreement with this type of scenario: the for comfort. Indeed, the subjects of our mutation loads ancestral population of Homo sapiens, having formed in and of our species preservation give rise to such grave Africa, came in prolonged contact with Neanderthal questions, especially with the spectres of eugenics and populations when it started colonising more northern Nazism still looming in our not so distant past, that I felt latitudes, and the two most probably competed for it was best to discuss the data and the situation of Homo territory occupation. Under such conditions, it would not Sapiens separately. be surprising if Haldane’s rule applied between Homo sapiens and Neanderthal, with interspecies mating

28/09/10 36 resulting in hybrid progeny comprised of sterile males resulting in further swelling of the populations and of the and fertile females, for whom further mating with Homo sizes of towns and cities. sapiens males would be the most effective way to Today, nearly 50% of the world population lives in produce offspring. Over successive generations, genomic major town and cities [107]. For western populations, DNA from those females would thus have entered the one can thus consider that the situation has become gene pool of the Homo sapiens population during its out progressively panmictic in just a few generations, as of Africa colonising migration , which could actually testified by the study of regions of extended have proven to be a very effective strategy to acquire sets homozygosity in samples of the North American of genes that were better adapted to the colder and population, which found that average inbreeding greener territories being colonised, and which the coefficients were above 1% in people born in 1900, but Neanderthal populations had inhabited, and thus adapted nearing zero in those born around 2000 [108]. These to for hundreds of thousands of years. changes in population structures are widely perceived as To date, despite this hybridisation with Neanderthal, beneficial because they should result in reduced and despite the fractionation of Homo sapiens into incidence in the occurrence of rare genetic diseases due separate races for tens of centuries, Homo sapiens is still to recessive mutations [107, 109]. But, as has been clearly a single species because no population has been discussed at length in this essay, this could to be a very described that would be less fertile with another, or that short sighted perspective because it equates to, as Muller would differ in its overall genetic constitution, for once put it, “eating all of our cake today” by allowing the example a fixated chromosomal rearrangement. recessive mutation load to increase progressively to higher levels, until the rate of elimination by genetic Regarding the occurrence of inbreeding in humans, defects once again balances the rate of their there has been a considerable evolution over the past few accumulation [13]. decades. For many centuries, the structures of human populations were probably quite similar to those seen in Incest, the union of individuals related in direct line, great apes today, being split into groups of a few dozens, and thus sharing at least 0.25 of their genome, is avoided with some individuals, most often females, passing from and condemned as taboo in virtually all societies, and one group to another. Over the centuries, the advent of this situation probably evolved to counter our natural civilisation resulted in the progressive increase in the instincts attracting us to our closest kin, of which the size of those groups, driven by a whole range of reasons, famous Oedipus complex is probably the most striking among which the most significant were probably i) the example. Historically, consanguineous unions have been conflicts with adjacent groups (with the smaller groups particularly prominent in rural populations as well as in being eliminated) ii) the advent of agriculture, which the upper classes (for example among Egyptians imposed sedentarity and allowed the sustenance of pharaohs or European royalty and aristocracy), whilst denser populations iii) the specialisation of individuals stern avoidance of consanguinity is mostly a trait typical into classes of farmers, craftsmen, soldiers, carers … of more urban middle classes. Today, the attitudes of resulting in an increase in the groups’ critical mass, i.e. various societies and cultures towards consanguinity the number of people necessary for having sufficient diverge greatly. Indeed, although first cousin marriage numbers of the various kinds in each group. are widely perceived as undesirable in the most Until the middle ages, the size of most human developed nations, and are even illegal in 31 or the 50

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 communities remained small, and average inbreeding states of the USA, as well as in China, this is not the case coefficients in human populations must thus have been in many other parts of the world such as the middle east quite significant. In this regard, the recent sequencing of or Asia, where weddings between uncles and nieces the genome from the hair of a 4000 year old Eskimo (degree of consanguinity F=0,25, resulting in offspring gave results consistent with an inbreeding coefficient of with an inbreeding coefficient I=0.125) or between first 0,06 [106], equivalent to that of the offspring of parents cousins ( F=0.125, I=0.06) are common, and even with 0.12 of consanguinity corresponding to the degree sometimes actively encouraged [109, 110]. Today, shared by first cousins. despite the phenomenal increase of the proportion of the Later on, recognising the existence of infectious human population living in urban environments, more microbes, leading to the concept of hygiene, did than 10% of the world’s unions are still consanguineous, considerably favour the increase in size of cities by and this is sometimes transiently reinforced in the decreasing the incidence of epidemics (when visiting the communities of recent urban immigrants [109, 110]. tower of London a few years ago, I learnt from the guide Although consanguineous marriages do result in a that, if London was the largest city in the world for many detectable cost in the fitness and viability of the years, it was thought to be related to the English’s love offspring, this is balanced by various factors such as of tea. Indeed, boiling the water greatly reduced the more stable marriages, better relationships between the spreading of water-born pathogens such a typhus, members of the extended family, a stronger sense of dysentery or cholera). The concept of aseptia also greatly community, enhanced female autonomy, and, reduced the numbers of deaths during childbirth. Later importantly, the economic benefits of keeping the family on, progress in medicine such as vaccination, antibiotics, land and belongings together [110]. Fifteen years ago, surgery would increase the survival of individuals, Bittles and Neel used a meta-analysis of 23 different

28/09/10 37 studies to compare the fate of the offspring from unions the differences in fertility rates between the wealthy and between first cousins with those from non- poor populations, even if the progress of molecular consanguineous parents, and estimated that first cousin biology will probably be able to help control genetic marriages resulted approximately in an additional 4% of loads by pre-natal screening, one does not need to be the offspring dying in the interval between 6 months called Thomas Malthus to see that the situation does gestation and ten years of age 15[111]. More recently, a indeed look poised for a progressive replacement of the study based on the complete birth records of the populations descended from those living today in more Icelandic population over the past 200 years not only developed countries by those coming from less confirmed that an evolution towards less consanguinity developed countries. This may be even amplified further was also taking place in Iceland, but also showed that by the well know fact that, when the standards of living couples that were consanguineous at the level of third or first increase in poor populations, this causes the fertility fourth cousins produced more grandchildren than those rates first to increase even more for one or two that were either more or less related [112]. The couples generations, before decreasing dramatically. that were more closely related had produced at least as many children, but a higher proportion of those had died The challenge for future generations will be to find a earlier and/or never reproduced, most probably as a model of society which, at the same time would provide consequence of deleterious recessive mutations. sufficient levels of quality of life to all human beings to curb their fertility, so that economies can be built on Today, the situation of human populations is clearly sustainable resources, and also promote the right balance not in a state of equilibrium, but in the process of between inbreeding and outbreeding: i) enough evolving rapidly. On the one hand, the populations of consanguinity to maintain mutation loads in check, and well developed countries combine low fertility rates with to nurture the perpetration of traditions and cultures, as panmictic reproductive strategies that will result in well as cooperative behaviours iii) enough outbreeding to significant increases in mutation loads for the future allow the shuffling of races, ideas and cultures. Indeed, generations, as well as promoting more and more selfish for ideas and for genes alike, exchanging and mixing is behaviours. On the other hand, the world’s most prolific the most effective way to promote the new encounters, populations are also the poorest (fertility rates are highest the new combinations that result in truly significant in Latin America, Sub-Saharan Africa and the Middle innovations and progress, i.e. true evolution. It is, at the East), and in many of those, the common occurrence of same time, also the best way to prevent the phenomenon consanguineous unions should maintain the mutation of speciation. For, even if it results in the awesome load to low levels, but this will presumably promote natural diversity that surrounds us today, I contend that further separation between the various populations of the the phenomenon of speciation first and foremost world in the long run. Indeed, although consanguinity corresponds to a loss in opportunities for exchange of rarely results in very high degrees of inbreeding in genetics materials between organisms, which is a direct humans, I contend that it is only a question of time consequence of the fact that, most of the time, it is before a significant chromosomal rearrangement finds initiated by the loss of a pre-existing function rather than itself associated to an advantageous recessive mutation. by the gain of a new one. If such a mutation were to become fixed in an certain portion of the population, which would then have Concluding remarks:

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 reduced fertility with the rest of the population, the questions of one or more species within the human race The ideas developed in this essay are mostly based on would become very real, and lead to extremely serious rather basic, not to say simplistic, concepts. One of the ethical concerns. reasons that kept me from writing up those ideas for Although circumstances such as wars, water rises due several years was the reasoning that, if this model was to global warming and food shortages due to even partially correct, then one of the many geneticists overpopulation represent much more pressing threats that have pondered about speciation for the past 150 today than those based on genetic events, the same may years should have developed similar ideas before me. not be true for the evolution of the balance between Today, I contend that, if similar ideas have not been various populations over the next coming decades. Given developed before, it is mostly because speciation has been considered as a phenomenon that should be 15 explained by population genetics. Most of the grounds Based on the figure of an increase of 4% in the for population genetics were, however, laid during the incidence of deaths between 6 months of gestation and first half of the twentieth century, initially by Fisher, 10 years of age in the offspring of first cousins, Beetles Haldane, and Wright with highly mathematical papers, & Neel concluded that the average mutation load must be and later by others such as Dobzhansky, Mayr and 0.7 lethal equivalent per gamete, and hence 1.4 per Muller, to reach the global concept of what is known as zygote. Considering that a large proportion of recessive “The modern synthesis”. All this groundwork by very mutations would probably provoke undetected early intelligent and gifted people took place before the abortions, we can presume that the average total load in structure of DNA, the genetic code, the digital nature of recessive deleterious mutations was at least twice that genetic information and the structure of genes were figure, and quite possibly somewhere between 5 and 10.

28/09/10 38 discovered, which all happened after WW2. But because selected against later on, and consequently so many they did not have access to this molecular knowledge, species around us. pre-war geneticists, and Muller in particular [13], considered all mutations as essentially dominant in their calculations. We now know that dominant mutations are Acknowledgements: usually due to a gain of function, recessive ones to a loss I am employed by INSERM, and I work at the IPBS, of function, and co-dominant ones to either a change of which depends both from CNRS and the University of function or to the effect of gene dosage. And we now Toulouse. I am particularly grateful to Fernando Roch, also know that most recessive mutations are indeed truly Patrick Bateson, Leigh van Valen, Chris Grobbelaar, recessive : having just one functional copy of a particular Tanguy Chouard, Pascal Gagneux, Philippe Druet and gene is usually sufficient, and heterozygotes with one Gilbert Fournié for the reading of earlier versions of this wild type and one mutated copy of a particular gene have manuscript, and for their comments and suggestions that absolutely no detectable phenotype, and a perfect have helped me to improve it. I am also extremely capacity to reproduce (contrarily to the pre-war grateful to the people who run the Google Scholar and assumption of an average effect of 2-5% effect on fitness JSTOR online services. Without those, I could never [13, 113]). As long as one does not recognise that many have found and accessed the information and mutations are truly recessive, one simply cannot venture bibliography that allowed me to develop and mature the towards the idea that deleterious ones are driving the ideas exposed in this essay. I am also grateful to Michael absolute requirement for inbreeding, and advantageous Lynch, Claudia Ricci, Erica Rosenblum and Alan Bittles ones the initial steps of speciation. who sent me copies of their work and replied patiently to my often blunt enquiries. Another factor that could have contributed to certain geneticists not following the paths I followed in these pages may have been related to the darkness, the political ‘incorrectness’ of the conclusions that these paths lead to regarding our future, and particularly that of our westernised populations. As Winston Churchill said: Once in a while you will stumble upon the truth but most of us manage to pick ourselves up and hurry along as if nothing had happened. But, as a geneticist, one should not lose sight of the fact that nature, and particularly the process of natural selection, is not politically correct. Indeed, when one thinks of the survival of the fittest, one often fails to consider the darker side of natural selection and that the counter-balance of the “survival of the fittest” is the “death ( or disappearance) of the less fit”. As considered at length by both Darwin and Wallace in their respective works, most reproducing organisms in natural

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 populations produce many more than two offspring, and of those, most will not go on to breed and their genes will hence disappear forever. I thus contend that the concept of "mildly deleterious mutations" is a very anthropocentric concept that derives from a very well fed, wealthy, healthy and secure self centred perspective. For the vast majority of living organisms, including most human beings on this planet today, there is no such thing as mild natural selection. Under natural conditions, the struggle for existence, as outlined by Darwin himself, is a very tough one in natural populations where only one in ten, hundred or even thousand of conceived zygotes will become mature organisms that go on to produce offspring. A very recent paper looking at wild population of field crickets reported the very unexpected observation that only one in ten of sexually active adults actually yielded offspring the following year, and this was true for both males and females [114] If it were not the case, we would not see so much variation, so many new characters being selected for in the first place, and

28/09/10 39 Addendum one : Bdelloid Rotifers: A scandal about a homogenisation of the DNA sequences. In addition, I ratchet, or a ratchet about a presumed scandal ? envisage that Bdelloids may have very good DNA Bdelloid Rotifers, which have been dubbed an repair, but rather low faithfulness in DNA replication. “evolutionary scandal” by John Maynard Smith, are the Indeed, this later trait may be required to maintain some only known example of multi-cellular organisms for level of adaptability in those asexual organisms. The which there is absolutely no doubt that they reproduce coupled processes of relatively unfaithful DNA strictly asexually. They are minuscule females ( < replication, together with homogenisation triggered as a 1mm), who can lay several dozens of parthenogenetic result of reiterated DNA damage occurring during eggs in the course of their 40-day adult lifetime. desiccation, may thus be replacing sex as a mean to Bdelloids are found in freshwater and the geological keep some level of adaptability in Bdelloids, whilst record tells us that they have been around for at least 35 fighting off infectious pathogens and Muller's ratchet at million year. In this sense, they are clearly among the the same time. This is in fact exactly equivalent to the most long lived “taxonomic species” in existence (I lottery that is played by sexual reproduction with a specify taxonomic here since the biological species degree of inbreeding, by keeping only those individuals concept only applies to sexual organisms), and they can that have at least one good copy of each gene, and be found all around the world, testifying of the success eliminating the unlucky ones that get two copies of a of their reproductive strategy. The downside of this is, bad one. In the short term, this will result in reduced however, that they probably have extremely limited numbers of individuals recovering from desiccation. But capacities for evolution, since they have apparently not given the bdelloid's individual prolificacy, repopulating yielded any more elaborate descendants over that very their environment after a cycle of desiccation does long period. It therefore seems fair to say that they may presumably not represent a major challenge, whilst well be stuck in an evolutionary dead end, from which keeping their genome functional must be one ! This is more elaborate life forms are extremely unlikely to why I suggest that, in Bdelloids, the desiccation cycles arise. Outside of their asexual lifestyle, Bdelloids have would thus act in place of sexual reproduction to fight three very special peculiarities which, I contend, are off the accumulation of deleterious recessive mutations. related to the need to cleanse their diploid genome from And other classes of animals that undergo recessive mutations: anhydrobiosis, such as the tardigrades or the - They are only found in wet or moist habitats that are darwinulids, may also be taking advantage of prone to successive rounds of desiccation and desiccation for regular shearing of their DNA to ensure rehydration. This correlates with what is called homogenisation of their diploid genomes. anhydrobiosis, i.e. the capacity to survive complete Radiations induce damages to DNA that are very similar dehydration at any stage of their life cycle. to those caused by desiccation. In several places on our - Individuals kept under continuous state of hydration will planet, the use of nuclear power by humans has caused quite rapidly show reduced fitness compared to and still causes the natural environment to be exposed to individuals undergoing regular cycles of dehydration, very high levels of radiation. As a rather wild which maintain the level of fitness seen in the seeding prediction, I would not be surprised if certain asexual stock [115]. A very recent study suggests that the main forms of life turned out to be able to adapt to those reason for this reduced fitness is presumably due to environments, using radiations instead of the cycles of Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 pathogens such as parasitic yeasts, which the bdelloids desiccation used by the bdelloids, both to provoke are not armed to eliminate. During the desiccation intermittent haploidisation of some of their genome, and cycles, however, bdelloids will scatter randomly to to destroy any infectious pathogens. other locations, where the pathogens will not have followed them, and will then be able to resume their life cycle without the pathogens, at least for a while [116]. - They are the most radiation resistant organisms known to date [117], due to an amazing capacity to repair damages to their genomic DNA, which can be explained by the fact that during the dehydration which is part of their natural life cycle, DNA will sustain multiple damages and strand breakage. My interpretation of the observation that bdelloids are primarily founds in environments that are prone to regular desiccation rather than in permanently hydrated surroundings is that the desiccation cycles could act in place of sexual reproduction to fight off the accumulation of deleterious mutations. Indeed, after DNA has been extensively chopped up by desiccation, DNA repair will involve chromosomal pairing and gene conversion will presumably cause significant

28/09/10 40 Addendum 2 : Three particular examples of the both the mammals and the bird sex chromosomes [120, occurrence of haploidy in eukaryotes: 121]. Yet another possibility of sex chromosome How does it feel…. to be on your own ? Bob Dylan arrangements is for the males to carry just one copy of the sex chomosome (XO, in certain insects like - Haploid stages: Within the frame of the biological grasshoppers and roaches), or the females (ZO, in some species concept, the phenomenon of speciation is only butterflies), whilst the rest of their genomes is diploid. relevant to the organisms that can reproduce sexually, For all those species, the sex chromosomes they contain i.e. that can go through meiosis. Through the process of will be in a haploid state either all the time (Y and W meiosis, a diploid cell will become haploid by chromosomes ), or in half of the individuals (X in males eliminating half of its chromosomes, and later fuse with and Z in females). For the genes carried by these another haploid cell to restore a state of diploidy. A chromosomes, the accumulation of recessive mutations critical step in the process leading to meiosis is the will hence not be a particular problem. The selective pairing of chromosomes, during which many events of pressures that they are submitted to, and the rate at recombination occur such as crossing-overs and gene which these genes evolve has, indeed, been found to conversion, which ultimately contribute to differ quite significantly from the genes carried by homogenisation of sequences, and can influence the rate autosomes ( see [122] for recent review), and the recent of occurrence of mutations via processes such as biased comparison of the human and chimpanzee Y gene conversion [118]. Depending on the organisms, the chromosomes has revealed an unexpectedly high level haploid state can last for more or less time, and even of divergence between the two, both in sequence and implicate stages of haploid cell division. Certain classes structure [123]. As developed in section II, the haploid of organisms, such as yeasts, fungi, algi, many plants character of sexual chromosomes in heterogametic and social insects, systematically pass via haploid stages individuals could be a central factor in allowing during their life cycles. All these species hence go selective pressures to give rise to hybrid sterility in through the most thorough screen possible for those heterogametic individuals whilst remaining silent eliminating recessive deleterious mutations, and would in homogametic ones (Haldane’s rule [40]), thereby not need inbreeding to fight Muller’s ratchet. Social favouring the inbreeding that will ultimately lead to insects ( ants, bees, wasps and termites) are known as speciation. Another very interesting question relates to haplodiploids because the males are haploid, whilst the what evolutionary forces can drive the selection of females are diploid. An explanation for the fact that this systems involving males and females having different strategy has promoted their social behaviour is that, in roles in reproduction, and what advantages these various species where the queen mates with only one male, such systems may have compared to one another. I plan to as honey bees, the female workers are more related to come back to those questions at a later date, but for the the offspring of their mother (75%) than to any time being, I will simply limit myself to proposing that offspring they would produce themselves if they were to one of the reasons favouring the development of those mate ( 50%). Hence, at every generation, half of a social various systems implicating sexual chromosomes may insect’s genome goes through a haploid stage that must be the difference in selective pressures in males and give rise to a fully fit and sexually active male. The fact females on the genes carried by those chromosomes that haplodiploid insects do not require inbreeding for (such as, for an example different from sexually related the maintenance of their genome is supported by the characters, the opsin genes, which lead to frequent

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 fact that they actually have a safeguard against colour blindness in men, and very seldom in women). inbreeding: according to the complementary allele - Endosymbionts: Apart from a nuclear envelope, the model, the sex-determining locus of social insects must second characteristic feature of most eukaryotes is that be heterozygous for the generation of a female [119]. In they possess mitochondria, the powerhouses of haploid males, the locus is necessarily hemizygous. If eukaryotic cells, which provide ATP via respiration. inbreeding takes place, i.e. if the allele of the sex- Since Lynn Margulis proposed it in the late 60’s, it has determining locus carried by the male matches that of been globally accepted that the ancestral aerobic one of the two carried by the queen, half of the eggs will eukaryote arose as a symbiotic arrangement whereby an be homozygous at the sex-determining locus, and this aerobic bacteria, probably related to the rickettsia, was will give rise to males, but they will be infertile because engulfed by the anaerobic ancestor of eukaryotes, which their offspring would be triploid. probably helped it to cope with the levels of oxygen - Sex chromosomes: Although not all animal species which started rising 2.5 billion years ago due to the where males and females can be found have sexual appearance of photosynthetic cyanobacteria on our chromosomes, this is by far the most common situation. planet. This engulfed aerobic bacterium was the In such species, including ours, the genomes of males ancestor of the mitochondria found in the cytoplasms of and females differ in the chromosomal composition, virtually all eukaryotes today. On at least three separate with either the males being heterogametic (XY, as in occasions, photosynthetic eukaryotes would later arise mammals, or flies), or the females (ZW, as in certain by the engulfment of cyanobacteria by early eukaryotes, insects, fish, reptiles and birds). The platypus, the only giving rise respectively to the green, red and the known egg-laying mammal, carries as many as 10 sex glaucophytes’chloroplasts. One remarkable aspect chomosomes (5X and 5Y), which share features with regarding all these endosymbiotic organelles is that they

28/09/10 41 have been maintained as separate entities for billions of problem rather than an advantage because, for the years in the cytoplasm of their hosts, where they still harmonious development of multi-cellular organisms, if replicate by fission, similarly to their bacterial they had inherited a mixed pool of mutated and un- ancestors. And during all that time, although some of mutated mitochondria, they would end up loosing a their genes have ‘migrated’ to their hosts’ genomes, all significant portion of their cells in certain organs where those endosymbionts have maintained their own self- the mutated mitochondria would have randomly taken replicating circular genomes. Yet, in most species, the over. The final picture that delineates itself from this endosymbiotic organelles are inherited from only one type of reasoning underlines the close relationship that parent [124]. The fact that, in yeasts, sexual ties sex and the need to cleanse obligatory diploid reproduction results in bi-parental transmission of genomes off the recessive mutations that they tend to mitochondria argues in favour of the view that, when accumulate silently. sex evolved in the ancestral diploid eukaryote, both parents probably contributed to the offspring’s initial stocks of mitochondria. And at first glance, this may Addendum 3 : The social lifestyle of a lowly amoeba. seem like a very suitable solution, since having two Dictyostelium discoideum (Dd) is an amoeba, which is populations of mitochondria would effectively be found in the soil of forests, where it feeds on bacteria. equivalent to being diploid, and should hence favour On rare occasions, when Dds of different mating types adaptive evolution by promoting the occurrence of new find themselves growing side by side in conditions of gene combinations. The fact that bi-parental inheritance darkness and moderate abundance of nutrients, they of mitochondria has almost universally evolved into undergo sexual reproduction, which involves the uni-parental modes (mostly from the mother, but formation of a macrocyst [126]. Most of the time, sometimes also from the father) does, however, suggest however, Dd amoebas multiply asexually, by mitosis. that bi-parental inheritance of mitochondria must have When food becomes scarce, these unicellular had more disadvantages than advantages. The first eukaryotes, that were until then growing completely obvious problem would be that it would inevitably lead independently from one another, will gather to form a to Darwinian competition between the two stocks of microscopic slug that can then migrate towards the bacteria, and that the host could end up paying the price surface, and form a minuscule plant-like structure, with of this intestinal wrestling [125]. The second problem is a stalk and a spore-containing head. Of the 100.000 the one related to the subject being discussed here, i.e. cells that gathered at the start, around 60 to 70 % will the maintenance of the integrity of diploid genomes. end up as spores, with an increased chance of reaching Although the main role of mitochondria is respiration, more suitable environments. But this will be at the cost they are also endowed with many other functions such of 30 to 40 % of the initial stock having sacrificed their as regulation of cell potential, calcium signalling, chances of survival to differentiate in stalk cells, or apoptosis, and various metabolic pathways. If the stocks other cells types. In the lab, one can see that slugs will of mitochondria were systematically inherited from both form by incorporating amoebae that are not necessarily parents, they would effectively behave as diploids, and related to one another, and at first glance, this would recessive mutations in the genomes of some of them seem particularly prone to promote the evolution of could be tolerated because they would be complemented selfish behaviour, whereby some individuals would by the function of the others. This could not be fixed by avoid ever becoming stalk cells [127]. This can actually

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 recombination between the genomes of the be found under experimental conditions, where the mitochondria because they do no perform sexual amoebae are grown in bulk, but this is not what is seen reproduction, and hence recombine only rarely. But in the wild : Dictyostelium amoebae that are found in during mitosis of eukaryotic cells, mitochondria are forest soils are usually all prone to forming well passed onto daughter cells following simple passive proportioned fruiting bodies, with the optimal distribution. Over several divisions, many cells will proportion of cells sacrificing themselves towards the hence end up with only one type of mitochondria. This doomed stalk lineage. I contend that, if Dictyostelids would not necessarily be very serious for a mono- have been able to evolve this social lifestyle, it is cellular organism because those unlucky cells inheriting because of their capacity to sporulate and disseminate, just mutated mitochondria would simply die out and and hence for single individuals ( or at most a handful make more room for the others. In certain plants, of amoebae originating from the same fruiting body) to chloroplasts can be inherited from both parents. In such colonise new isolated niches. The resulting populations plants, it is possible to isolate variegated varieties, due must thus be comprised of groups of individuals that are to the fact that one of the parents carries mutant highly related to one another, or even very often clonal. chloroplasts that can no longer make chlorophyll. The Under such conditions, selfish mutants will be doomed variegations correspond to areas of the plants that have, because, when they find themselves on their own, their randomly, lost the chloroplasts that could make incapacity to form stalk cells will condemn the fate of chlorophyll. Such plants are, however, not found in their offspring to staying in the same spot. natural environments. For animal mitochondria, it is rather easy to picture how the inheritance of a diploid pool of mitochondria could rapidly become a significant

28/09/10 42 Addendum 4: Chromosomal translocations and saeptation

Here, I consider how chromosomal translocations could If the phenotype associated to the chromosomal fit within the types of scenario envisaged in section II of rearrangement is recessive, given the inherent the main text, i.e. at the very early stages of speciation. disadvantage of rearrangements due to reduced fertility, As discussed in that section, it seems very difficult to one would only ever expect a few heterozygotes to arise conceive that a chromosomal rearrangement could ever in the population since those would not benefit from the reach fixation within any population unless it was phenotype. Such mutations would therefore seem directly associated with an advantageous phenotype that unlikely to persist in the population for many would at least compensate the reduced fertility inherent generations, and revelation of the beneficial phenotype to that rearrangement. Many types of advantages could would thus occur mostly in individuals with very high be envisaged, but the two most likely would seem to be degrees of consanguinity. Furthermore, in the case of a either increased fitness (such as strength, sexual reciprocal translocation, only one in 6 of the viable attraction, resistance to harsh conditions) or adaptation offspring from two heterozygotes would be to a new environment, and that advantageous phenotype homozygous (corresponding to one out of 16 possible associated to the rearrangement could have either a zygotes, Fig. 1). This type of scenario would thus dominant, recessive, or co-dominant phenotype. We can presumably result in very tight population bottlenecks. thus repeat an analysis similar to that performed for Among the few individuals becoming homozygotes, recessive, dominant, or co-dominant (co-recessive) however, fixation of the rearrangement would be mutations, with the added dimension of the reduced immediate, and breeding with the ancestral stock would fertility conferred by the rearrangement itself. result in offspring with the double disadvantage of the If the rearrangement results in a fully dominant increase reduced fertility, and loss of the recessive advantageous in fitness, that trait will have a natural tendency to trait. spread in the population despite the reduction of fertility conferred by the chromosomal rearrangement, and could thus quite possibly reach fixation without actually undergoing bona fide saeptation. If the rearrangement results in a dominant trait that drives individuals to colonise a new environment, this will, as seen earlier, automatically result in some significant degree of inbreeding in the newly adapted group, which could favour fixation of the rearrangement, and promote further saeptation by promoting the surfacing of additional adaptive, but recessive mutations. If the phenotype is actually co-dominant (or co-recessive), with homozygous individuals being even fitter than heterozygotes, this may actually promote inbreeding to some degree, and thus a reduction of the gene flow

Nature Precedings : hdl:10101/npre.2010.5003.2 Posted 20 Oct 2010 towards the rest of the population. In addition, individuals becoming homozygous for the chromosomal rearrangement will have a further significant advantage in breeding with one another because this would eliminate the reduction of fertility due to chromosomal misassortments.

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